Wood Structure in Plant Biology and Ecology [1 ed.] 9789004265608, 9789004265592

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Wood Structure in Plant Biology and Ecology

Cover: Logo of the WSE meeting illuminating the woody macchia vegetation along the coast north of Naples, Italy. The island of Ischia is in the distance. By courtesy of Veronica De Micco.

Wood Structure in Plant Biology and Ecology edited by Pieter Baas Giovanna Battipaglia Veronica De Micco Frederic Lens Elisabeth Wheeler

IAWA Journal 34 (4), 2013 International Association of Wood Anatomists c/o Naturalis Biodiversity Center, Leiden, The Netherlands

Library of Congress Cataloging-in-Publication Data are available from the Publisher, Leiden, The Netherlands.

ISBN: 978 90 04 26559 2 © Copyright 2013 by Koninklijke Brill NV, Leiden, The Netherlands. Koninklijke Brill NV incorporates the imprints Brill, Global Oriental, Hotei Publishing, IDC Publishers and Martinus Nijhoff Publishers. All rights reserved. No part of this publication may be reproduced, translated, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the publisher. Authorization to photocopy items for internal personal use is granted by Brill provided that the appropriate fees are paid directly to Copyright Clearance Center, 222 Rosewood Drive, Suite 910, Danvers, MA 01923, USA. Fees are subject to change. Printed in The Netherlands.

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Contents Preface

P. Baas, G. Battipaglia. V. De Micco, F. Lens & E.A. Wheeler: Wood structure in Plant Biology and Ecology ...........................................................................................

page

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Reviews

C. Strullu-Derrien, P. Kenrick, E. Badel, H. Cochard & P. Tafforeau: An overview of the hydraulic systems in early land plants . ................................................................. T. Anfodillo, G. Petit & A. Crivellaro: Axial conduit widening in woody species: a still neglected anatomical pattern ................................................................................. S. Rosner: Hydraulic and biomechanical optimization in Norway spruce trunkwood – A review............................................................................................................... P. Prislan, K. Čufar, G. Koch, U. Schmitt & J. Gričar: Review of cellular and subcellular changes in cambium . .............................................................................................

5 24 37 63

New methods

C.R. Brodersen: Visualizing wood anatomy in three dimensions with high-resolution X-ray micro-tomography (µCT) – A review . ............................................................. L. Wegner, G. von Arx, U. Sass-Klaassen & B. Eilmann: ROXAS - an efficient and accurate tool to detect vessels in diffuse-porous species . ........................................ G. von Arx, C. Kueffer & P. Fonti: Quantifying plasticity in vessel grouping – added value from the image analysis tool ROXAS ...............................................................

80 97 105

Environmental effects on wood structure

V. De Micco, E. Zalloni, A. Balzano & G. Battipaglia: Fire influence on Pinus halepensis: wood responses close and far from scar . ....................................................... K. Novak, M. A. Saz Sánchez, K. Čufar, J. Raventós & M. de Luis: Age, climate and intra-annual density fluctuations in Pinus halepensis in Spain ............................... S. Stojnic, U. Sass-Klaassen, S. Orlovic, B. Matovic & B. Eilmann: Plastic growth response of European beech provenances to dry site conditions ............................ F. H. Schweingruber, L. Hellmann, W. Tegel, S. Braun, D. Nievergelt & U. Büntgen: Evaluating the wood anatomical and dendroecological potential of arctic dwarf shrub communities .......................................................................................................... M. S. Costa, T. J. de Vasconcellos, C.F. Barros & C.H. Callado: Does growth rhythm of a widespread species change in distinct growth sites? .........................................

118 131 147 157 170

The page numbers in the above Table of Contents refer to the bracketed page numbers in this volume.

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Wood Structure in Plant Biology and Ecology Pieter Baas 1, Giovanna Battipaglia 2, Veronica De Micco 3, Frederic Lens 1, and Elisabeth Wheeler 4 (editors) 1 Naturalis

Biodiversity Center, PO Box 9517Leiden, The Netherlands di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche (Di. S.T. A. Bi. F), Seconda Università di Napoli, via Vivaldi 43, I-81100 Caserta, Italy 3 Dipartimento di Agraria, Università degli Studi di Napoli Federico II, via Università 100, I-80055 Portici (NA), Italy 4 Department of Forest Biomaterials, NCSU, Raleigh, USA

2 Dipartimento

This special issue of the IAWA Journal contains original and review papers presented at the successful meeting of the International Association of Wood Anatomists (IAWA), convened in the framework of the International Symposium “Wood Structure in Plant Biology and Ecology”, held in Naples from 17–20 April 2013 and organized by the University of Naples Federico II and the Second University of Naples on behalf of the COST-Action STReESS, ‘Studying Tree Responses to extreme Events: a SynthesiS’. Xylem of trees, shrubs, and also herbs plays a crucial role in plant biology and ecology (Baas & Miller 1985). The evolutionary fitness of each plant species depends to a large extent on the fine balance between its hydraulic efficiency and safety, its cost-effective biomechanical design, and its biological defense, all in equilibrium with specific requirements posed by the physical and biological conditions of its environment. Moreover, wood constitutes a historical archive of the changing environmental and climate conditions as well as episodic or incidental stresses to which an individual tree or shrub has been exposed. This in turn allows studying plant responses to environmental change in past and present, and hypothesizing future effects of climate change. Almost 350 years after the microscopic structure and functions of wood were first discovered and hotly debated by the early microscopists Malpighi, Grew and Van Leeuwenhoek, the study of functional and ecological wood anatomy enjoys a renaissance and plays a pivotal role in plant and ecosystem biology, global change research and the understanding of plant evolution (Groover & Cronk 2013). The programme of the Naples meetings represented a very full and rich spectrum of all aspects of wood structure in plant biology and ecology. In a uniquely cooperative spirit, four international Journals (Dendrochronologia, IAWA Journal, Trees, and Tree Physiology) collaborated to publish as many of the high quality papers presented at the meeting as possible. Dendrochronologia will publish a selection of tree-ring studies; Trees and Tree Physiology will publish some of the papers with a more strictly © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000028

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(eco)physiological focus. The IAWA Journal is proud to be the first to dedicate a full special issue to the proceedings of this meeting, including papers with an anatomical focus. The selected papers give a good cross section of current developments in the study of functional and ecological wood anatomy. Review papers address the hydraulic architecture of the earliest woody land plants (Strullu-Derrien et al.), the general and functionally crucial phenomenon of axial conduit widening (Anfodillo et al.), the hydraulic and biomechanical optimization in a very important timber species (Rosner), and cellular and subcellular changes in the cambium in response to environmental factors (Prislan et al.). New tools to study structural aspects of hydraulic functioning of woody plants are reviewed in a paper on high-resolution 3-D visualization of wood structure (Brodersen), and applications of the ROXAS software to vessel detection and vessel grouping (papers by Wegner et al. and von Arx et al.). About half of the papers represent a bouquet of case histories in wood studies as applied to plant biology and ecology: on the effects of fire on wood structure (De Micco et al.) and on the occurrence of intra-annual density fluctuations in response to late summer/early autumn rains (Novak et al.), both in Aleppo pine. Stojnic et al. report on plastic growth and anatomical responses in beech. Two papers not presented in Naples, but submitted in recent months to the IAWA Journal also fit the theme of this special issue: a paper on changing growth rhythms in a widespread deciduous tropical hardwood (Costa et al.) and an overview of wood anatomical variation in ten common arctic dwarf shrub species (Schweingruber et al.). Altogether an inspiring collection of contemporary applications of wood anatomy to better understand the ecophysiology of woody plants. Apart from the normal print-run as IAWA Journal 34 (4), Brill is also publishing a trade edition of this special issue. Editors and Guest Editors (listed here alphabetically) wish to thank all authors and anonymous reviewers for submitting manuscripts, reviews and revisions under considerable time pressure. We also wish to acknowledge the financial and logistic support of the meeting by the Cost-Action STReESS and its very cooperative Chair, Dr. Ute Sass-Klaassen. References Anfodillo T, Petit G & Crivellaro A. 2013. Axial conduit widening in woody species: a still neglected anatomical pattern. IAWA J. 34: 352–364. Baas P & Miller RB. 1985. Functional and ecological wood anatomy – some introductory comments. IAWA Bull. n.s. 6: 281–282 (in special issue dedicated to proceedings of the Martin H. Zimmermann memorial symposium). Brodersen CR. 2013. Visualizing wood anatomy in three dimensions with high-resolution X-ray micro-tomography (µCT) – A review. IAWA J. 34: 408–424. Costa MS, de Vasconcellos TJ, Barros CF & Callado CH. 2013, Does growth rhythm of a widespread species change in distinct growth sites? IAWA J. 34: 498–509.

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De Micco V, Zalloni E, Balzano A &Battipaglia G. 2013. Fire influence on Pinus halepensis: wood responses close and far from scar. IAWA J. 34: 446–458. Groover A & Cronk Q. 2013. From Nehemiah Grew to genomics: the emerging field of evo-devo research for woody plants. Int. J. Plant Sci. 17: 959–963. Novak K, Saz Sánchez MA, Čufar K, Raventós J & de Luis M. 2013. Age, climate and intraannual density fluctuations in Pinus halepensis in Spain. IAWA J. 34: 459–474. Prislan P, Čufar K, Koch G, Schmitt U & Gričar J. 2013. Review of cellular and subcellular changes in cambium. IAWA J. 34: 391– 407. Rosner S. 2013. Hydraulic and biomechanical optimization in Norway spruce trunkwood – A review. IAWA J. 34: 365–390. Schweingruber FH, Hellmann L, Tegel W, Braun S, Nievergelt D & Büntgen U. 2013. Evaluating the wood anatomical and dendroecological potential of arctic dwarf shrub communities. IAWA J. 34: 485– 497. Stojnic S, Sass-Klaassen U, Orlovic S, Matovic B & Eilmann B. 2013. Plastic growth response of European beech provenances to dry site conditions. IAWA J. 34: 475–484. Strullu-Derrien C, Kenrick P, Badel E, Cochard H & Tafforeau P. 2013. An overview of the hydraulic systems in early land plants. IAWA J. 34: 333–351. von Arx G, Kueffer C & Fonti P. 2013. Quantifying plasticity in vessel grouping – added value from the image analysis tool ROXAS. IAWA J. 34: 433–445. Wegner L, von Arx G, Sass-Klaassen U & Eilmann B. 2013. ROXAS - an efficient and accurate tool to detect vessels in diffuse-porous species. IAWA J. 34: 425–432.

Citation Chapters in this book should be cited as regular papers in IAWA Journal 34(4), using the high page numbers indicated on the title pages.

Journal 342013: (4), 2013 IAWAIAWA Journal 34 (4), 333–351

An overview of the hydraulic systems in early land plants Christine Strullu-Derrien1,*, Paul Kenrick1, Eric Badel 2,3, Hervé Cochard 2,3 and Paul Tafforeau 4 1Department

of Earth Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom 2INRA, UMR547 PIAF, 63100 Clermont-Ferrand, France 3 Clermont Université, Université Blaise Pascal, UMR547 PIAF, 63000 Clermont-Ferrand, France 4 European Synchrotron Radiation Facility, 6 rue Jules Horowitz, 38043 Grenoble cedex, France *Corresponding author; e-mail: [email protected]

abstract

One of the key functions of wood is hydraulic conductivity, and the general physical properties controlling this are well characterized in living plants. Modern species capture only a fraction of the known diversity of wood, which is well preserved in a fossil record that extends back over 400 million years to the origin of the vascular plants. Early fossil woods are known to differ in many key respects from woods of modern gymnosperms (e.g., tracheid size, secondary wall thickenings, lignin chemistry, cambium development) and recent discoveries are shedding new light on the earliest stages of wood evolution, raising questions about the performance of these systems and their functions. We provide an overview of the early fossil record focusing on tracheid morphology in the earliest primary and secondary xylem and on cambial development. The fossil record clearly shows that wood evolved in small stature plants prior to the evolution of a distinctive leaf-stem-root organography. The hydraulic properties of fossil woods cannot be measured directly, but with the development of mathematical models it is becoming increasingly feasible to make inferences and quantify performance, enabling comparison with modern woods. Perhaps the most difficult aspect of hydraulic conductance to quantify is the resistance of pits and other highly distinctive and unique secondary wall features in the earliest tracheids. New analytical methods, in particular X-ray synchrotron microtomography (PPC-SRμCT), open up the possibility of creating dynamic, three-dimensional models of permineralized woods facilitating the analysis of hydraulic and biomechanical properties. Keywords: Wood, fossil, synchrotron, 3D model, permineralization, hydraulic properties. Introduction

The evolution of the vascular system in plants was a key development in the history of life because of its fundamental role in water transport and, in many species, its ancillary function as a framework of structural support (Sperry 2003; Pittermann 2010; Lucas © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000029

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IAWA Journal 34 (4), 2013 Monilophytes Lignophytes

Sil 443

Ord

Spermatophytes

Archaeopteridales

Aneurophytales

Filicophytes

Sphenophytes

Cladoxylopsids

Lycopsids

Euphyllophytes

Psilophyton

L

Cooksonia

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Coleochaetales

U

Dev M

Rhyniophytes

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Bryophytes

Charales

Car Mis

Anthocerophytes

Pen

Marchiantiophytes

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Zosterophyllopsids

Lycophytes

Bryophytes

Tracheophytes Embryophytes

488 C-Type tracheids

S-Type tracheids

Secondary xylem, unifacial cambium

G-Type tracheids

P-Type tracheids

Secondary xylem, bifacial cambium

Figure 1. Simplified phylogenetic tree showing the minimum stratigraphic ranges of selected groups based on megafossils (bars) and their minimum implied range extensions (lines). Tracheid and secondary xylem types are shown on the figure. Ord = Ordovician; Sil = Silurian; Dev = Devonian, L = Lower, M = Middle, U = Upper; Car = Carboniferous. Mis = Mississippian, Pen = Pennsylvanian. Adapted from Kenrick & Crane (1997b).

et al. 2013). The vascular plants or tracheophytes are defined by the possession of this tissue system, the acquisition of which was essential to the evolution of their diverse forms, leading ultimately to their dominance of terrestrial ecosystems (Niklas 1997; Bateman et al. 1998; Labandeira 2005). The main constituents of the vascular system are phloem and xylem, but it is the latter that is more commonly encountered in the fossil record due to the resilience of its cellular components, which typically possess robust cell walls containing the polyphenolic polymer lignin (Boyce et al. 2004). Vascular tissues first appear in the fossil record in the lower part of the Devonian Period (410– 407 Myr) (Fig. 1) when terrestrial sediments containing fossil plants first became abundant (Gensel 2008; Kenrick et al. 2012). Research over the past 25 years has revealed some of the earliest stages in the evolution of the xylem (Stein 1993) and in particular has focused on the interpretation and documentation of its main component, the tracheid (Kenrick & Crane 1991; Edwards 1993; Friedman & Cook 2000; Sperry 2003). Several distinctive types of tracheid are now widely recognized, providing detailed information on the structure of the cell wall and insights into the early evolution of this important cell type. The vascular system in the earliest tracheophytes was entirely primary, but the fossil record has also provided much information on the evolution of secondary vascular tissue (Cichan 1986; Cichan & Taylor 1990; Meyer-Berthaud et al. 2010; Spicer & Groover 2010; Gerrienne et al. 2011). Cambial activity in plants initially evolved indepen-

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dently at least twice in the Devonian Period (Kenrick & Crane 1997a, b) and perhaps on more occasions (Boyce 2010) (Fig. 1). In many extinct groups the cambium was unifacial, developing only secondary xylem, and it was unable to undergo anticlinal cell division (Taylor et al. 2009; Spicer & Groover 2010). This type of cambium produced the secondary xylem in the extinct tree cladoxylopsids of the Devonian Period and the lycopods and horsetails of the Carboniferous Period (Niklas 1997; Stein et al. 2007; Meyer-Berthaud et al. 2010). These trees generally produced rather little wood, and the vascular cylinder had limited capacity to increase in volume while maintaining the integrity of the cambium (Cichan 1986). The more familiar bifacial cambium gives rise to both secondary xylem and secondary phloem, and it has the capacity to undergo anticlinal cell division (Donoghue 2005). This form of cambium evolved during the Devonian Period in the lineage leading to gymnosperms and angiosperms (Hilton & Bateman 2006). New information from the early fossil record is providing further tantalizing insights that are promising to unravel the sequence of acquisition of characteristics that led to the evolution of secondary xylem (Gerrienne et al. 2011). Our growing knowledge of the early evolution of vascular tissues derived from the careful study of fossils raises questions about the performance of these systems and their functional roles in the early development of vascular plants. Recent advances in our understanding of the hydraulics of modern woods (Hacke et al. 2004; Pittermann et al. 2006; Pittermann 2010) and the development and application of mathematical methods to infer the hydraulic properties of fossil woods (Wilson et al. 2008; Wilson & Knoll 2010; Wilson & Fischer 2011) mean that we now have many of the tools that we need to begin to investigate the hydraulic characteristics of the earliest vascular systems in plants. Early fossil wood is typically permineralized, and various methods that have been developed to investigate and characterize its structure include the preparation of sections for light microscopy and scanning electron microscopy (Jones & Rowe 1999). These are invasive, and they are frequently also destructive. The development of X-ray computed tomography, in particular the use of high-resolution tools such as synchrotrons, provides an efficient non-destructive alternative (e.g., Friis et al. 2007). Here we give a succinct overview of the earliest fossil record of primary and secondary xylem and its cellular components and an introduction to recent research on its hydraulic properties. We show how synchrotron microtomography can be used to investigate the hydraulic properties of the earliest wood. The earliest fossil evidence of vascular elements and their hydraulic structures

Xylem and tracheid cell structure are frequently well preserved in the fossil record enabling both biomechanical properties and hydraulic efficiency to be estimated, furthering our understanding of the functional evolution of wood (Niklas 1997; Sperry 2003; Pittermann 2010). In fossils of the early part of the Devonian period the vascular system typically is permineralized in a variety of minerals including pyrite and its oxidation products (Kenrick 1999) (Fig. 2c–e), more rarely silicates (Channing & Edwards 2009), calcium /magnesium carbonates (Hartman & Banks 1980), or is simply preserved as

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b

Figure 2. – a: Cooksonia pertoni: the earliest tracheophyte (Herefordshire, England; specimen nr. V58010 Lang collection, Natural History Museum, London). Scale bar = 0.15 mm. – b: Coalified tracheid in Cooksonia pertoni (C-type tracheid). Note the thick coalified wall and mineral infill of lumen with grooves marking positions of secondary thickenings (From Edwards, New Phytologist, 1993; with courtesy). Scale bar = 0.8 μm. – c–e: Highly polished transverse section through a pyritized axis of Gosslingia breconensis. – c: Whole axis showing a central elliptical xylem surrounded by an amorphous acellular area of pyrite (white) and an outer cellular area. Scale bar = 250 μm. – d: Higher magnification of the xylem strand. Note the presence of wall pyrite between the coalified (black) walls of adjacent cells. Scale bar = 100 μm. – e: Transverse section through the cells at the edge of the xylem (G-type tracheid) showing components of the tracheid wall. Note the continuous organic wall of the annular or spiral wall sculpture (*) and the broken organic wall that represents a perforate wall (arrowhead) lying between the wall thickenings. Scale bar = 25 μm.

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carbonized or charcoalified cells without associated mineralization (Edwards et al. 1992; Edwards 1993) (Fig. 2a, b). Interpreting cell wall structure is not straightforward, and the effects of decay and mineralization need to be critically evaluated when reconstructing tracheid characteristics and cell wall components (Kenrick & Edwards 1988; Kenrick & Crane 1991) (Fig. 3g–l). Some of the earliest plants possessed conducting systems comprising cells without the wall thickenings that characterize tracheids (Edwards 1993). By this we mean completely lacking helical or annular bars and pitting in the cell wall. One of the exceptionally well-preserved silicified plants from the 407-millionyear-old Rhynie Chert (Aglaophyton major) possessed a conducting system that strongly resembles the basic organization of leptoids (specialized food-conducting cells) and hydroids (specialized water-conducting cells) observed in some of the larger modern mosses (Edwards 1993), but phylogenetic analysis shows that this fossil is more closely related to the vascular plants than to bryophytes (Kenrick & Crane 1997a, b). Tracheids may therefore have evolved from hydroid-like antecedents. Early fossil vascular plants (Fig. 3a–f) possessed types of tracheid (Fig. 3g–l) that differ in significant ways from those of their modern relatives. One distinctive form is the S-type tracheid (Kenrick et al. 1991). This cell has large helical thickenings with a spongy interior (Fig. 3g, j). The lumen-facing surface is lined with a thin microperforate wall. Perforations within the wall measure c. 40 nm to 200 nm in diameter with a density of c.16 μm-2. The S-type tracheid has been observed in several stem group vascular plants (Rhynia gwynne-vaughanii, Sennicaulis hippocrepiformis, Huvenia kleui, Stockmansella sp.) (Fig. 1; 3a, d) in both their sporophyte and probably also their gametophyte generations and in two different forms of permineralization (i.e., pyrite, silicates; Kenrick & Crane 1991). The G-type tracheid is a second distinctive form that is widespread in stem group lycopods (Fig. 1; 3b, e). The cell is characterized by annular or helical thickenings with some cross connections (Fig. 3h, k). Typically, a distinctive perforate sheet of material occupies the cell wall between the thickenings (Kenrick & Edwards 1988), but the thickenings themselves are known to be perforate in one species (Wang et al. 2003). Perforations in this layer are typically an order of magnitude larger than those in the S-type cell and of less regular shape (Kenrick & Edwards 1988). The wall thickenings of both S-type and G-type cells are essentially helical or annular, but the extensive development of cross connections between bars can lead to simple reticulate pitting in the G-type cell (Kenrick & Crane 1997a, b). Bordered pitting developed in other tracheid types. This was an early innovation in vascular plants that evolved independently at least twice: once in lycophytes and once in euphyllophytes (Kenrick & Crane 1997a, b). Bordered pitting characterizes the P-type tracheid, which is common to many basal euphyllophytes (Fig. 1; 3c, f). Pitting is mostly of the scalariform type, and a distinctive feature is the presence of an additional perforate sheet of wall material extending over the pit apertures (Fig. 3i, l). The perforations in this sheet are distributed either in one of two transverse rows or less regularly in a reticulum (Hartman & Banks 1980; Kenrick & Crane 1997a, b). Within the lycophytes, a broadly similar pit construction is seen in the fossil Minarodendron, but the scalariform bars are much more elongate (Li 1990). These are slightly different to the P-type cell. In Minarodendron, the additional perforate sheet of wall material

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a



g

h

b

i

j

c

k

l

Figure 3. Early land plants and the main tracheid types. — a–c: Reconstructions of the plants. – a: Rhynia gwynne-vaughanii (from Kenrick & Crane 1997a). Scale bar = 1 cm. – b: Asteroxylon mackiei (from Kidston & Lang 1920). Scale bar = 1.5 cm. – c: Psilophyton dawsonii (from Kenrick & Crane 1997a). Scale bar = 1 cm. — d–f: Transverse sections of the axes. – d: Rhynia gwynne-vaughanii (slide nr. 3133, Scott collection, Natural History Museum, London). Scale bar = 0.6 mm. – e: Asteroxylon mackiei (slide nr. 1015, Lang collection, Natural History Museum, London). Scale bar = 2.5 mm. – f: Psilophyton dawsonii (slide nr. OH55 x.2, Florin collection, Naturhistoriska riksmuseet, Stockholm). Scale bar = 0.6 mm. — g–l: Diversity of tracheids in early land plants (median longitudinal section through the cells, the basal and proximal end walls are not shown; cells are 20– 40 μm in diameter). – g: S-type tracheid. – h: G-type tracheid. – i: P-type tracheid. – j: details of S-type cell wall showing ‘spongy’ interior to thickenings and distribution of perforations in thin lumen-facing layer. – k: details of G-type cell wall showing perforations distributed between thickenings. – l: details of P-type cell wall showing pit chambers and layer with perforations that extends over pit apertures.

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is attached to the pit aperture crossing the pit chamber, whereas in the P-type cell of Psilophyton it is positioned within the pit and closer to the pit-closing membrane. This additional perforate sheet of secondary wall material associated with pitting does not occur in modern groups, and it appears to be relatively short-lived in geological terms. Within euphyllophytes and many lycopods it is soon supplanted in the Devonian Period by tracheids with more conventional bordered pits. The feature persists in some arborescent lycopods (Cichan et al. 1981) into the Carboniferous Period. The functional significance of the additional perforate sheet crossing pit chambers is unknown, but we suggest that it would have increased the hydraulic resistance of the cell wall, reducing the risk of cavitation. The three types of early vascular element discussed above are widely recognized and have been characterized in some detail, but other variants are known to exist (e.g., C-type; Edwards 1993) (Fig. 2a, b). By the middle part of the Devonian Period tracheids with more conventional bordered pits without the additional perforate sheets of wall material had evolved (e.g., Leclercqia; Grierson 1976). In early vascular plants, the metaxylem is generally thought to be composed primarily of one or other of the main tracheid types. Thus, at any given point in a plant, the entire metaxylem would be composed of either the S-type, G-type or P-type tracheids. The assumption that one tracheid type would characterize a whole plant has recently been refuted with the discovery of G-type tracheids at one level and a pitted type at a more distal level within the same individual (Edwards et al. 2006). Thus, tracheid wall structure may vary quite significantly at different levels within an individual. The major features of the secondary walls of the earliest tracheids (i.e., presence or absence of thickenings and pitting) are distinctive and readily recognizable. One other common characteristic is the distribution pattern of organics and minerals observed within the permineralized cell walls. The significance of this needs to be interpreted carefully and with reference to the taphonomic processes involved (Kenrick & Crane 1991). In certain forms of permineralization, notably pyrite and its oxide derivatives, the partitioning of mineral and organics (Fig. 2e) most likely reflects the distribution of resistant polyphenolics (i.e., lignin) within the basic cellulose framework of the cell wall (Kenrick & Edwards 1988). Under this interpretation, the coalified parts of the wall are decay resistant and reflect the original pattern of lignification, whereas the mineralized parts represent areas that underwent substantial decay and are therefore likely to have been weakly lignified or non-lignified. This hypothesis has been tested through a detailed analysis of tracheid development and subsequent decay in the living lycopod Huperzia. Friedman and Cook (2000) showed that patterns of lignification in the tracheids of Huperzia broadly reflect those hypothesized in the early fossil tracheids. The fossil record shows that lignification of the cell wall in the earliest vascular plants was most well developed on the inner lumen-facing surface, but the thickness of this layer varied among cell types (Friedman & Cook 2000). In all cells that possess this feature, the inner lumen-facing lignified layer was perforate (Fig. 3j–l). The perforations were most numerous and smallest in the thin lignified wall of the S-type cell (Fig. 3j). Here, their size and distribution indicate that they might be plasmodesmata derived, because they share similarities to pores in the water-conducting cells of some living

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Figure 4. The three oldest (Early Devonian) euphyllophytes exhibiting wood. Transverse section of the xylem. – a: The plant from Châteaupanne (Armorican Massif, France). Scale bar = 200 μm. – b: The plant from New Brunswick (Canada) (photo P. Gensel, with courtesy). Scale bar = 300 μm. – c: Franhueberia gerrienni from Gaspé (Canada) (photo M. Tomescu, with courtesy). Scale bar = 200 μm.

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liverworts (e.g., Ligrone & Duckett 1996). The perforations were larger and typically more restricted in distribution in the thicker walls of the G-type tracheids (Fig. 3k), and they reached their most developed form in the bordered pit (Fig. 3l). As with bordered pits, from a functional perspective it is probable that the various forms of perforations in the walls of these early tracheids were channels enabling the flow of fluids through an otherwise impermeable wall layer. The earliest evidence of wood

The first forest ecosystems evolved by the middle part of the Devonian Period, and they were populated by arborescent plants belonging to several major lineages (Bateman et al. 1998; Gensel & Edwards 2001; Stein et al. 2007; Gensel 2008; Meyer-Berthaud et al. 2010; Cornet et al. 2012; Stein et al. 2012; Giesen & Berry 2013). Arborescence is known to have evolved in plants independently in many different groups, and a variety of biomechanical strategies were employed (Mosbrugger 1990; Niklas 1997; Rothwell et al. 2008; Pittermann 2010). In gymnosperms, the evolution of wood was key to the development of shrubs and trees, whereas it played a lesser role in the evolution of arborescence in several other groups of plants (Niklas 1997; Donoghue 2005; Meyer-Berthaud et al. 2010). The wood of early gymnosperms was complex, derived from a bifacial cambium which, through periclinal cell division, gave rise to secondary xylem towards the centre and secondary phloem towards the outside. The secondary xylem contained both tracheids and rays. The bifacial cambium was also able to undergo anticlinal cell division to accommodate increasing girth (Niklas 1997; Spicer & Groover 2010). Other early woods in the extinct arborescent cladoxylopsids, lycophytes and sphenophytes differed in some important features. With the possible exception of Sphenophyllum (Eggert & Gaunt 1973; Cichan & Taylor 1982), their cambia are generally thought to have been unifacial, producing only secondary xylem (Niklas 1997; Spicer & Groover 2010). Furthermore, in many but not all early woody plants the cells of the unifacial cambium were unable to divide anticlinally to produce new cambial initials. Thus, the cambium in these plants had limited capacity to increase in circumference and retain its integrity as girth increased (Donoghue 2005; Spicer & Groover 2010). Early fossils therefore show that the suite of characteristics that comprise wood in modern gymnosperms assembled over a period of time, with the capacity to produce secondary xylem appearing prior to the evolution of secondary phloem, and periclinal cell division of cambial initials appearing before the ability to sustain anticlinal divisions indefinitely. Recent research on fossils from the early part of the Devonian Period is beginning to shed further light on the earliest stages in the evolution of wood. The earliest vascular plants possessed entirely primary growth, but among euphyllophytes one occasionally sees short files of radially aligned xylem, giving the impression of secondary growth. This was observed in Lower Devonian fossils such as Psilophyton dawsonii (Banks et al. 1975) and Psilophyton crenulatum (Doran 1980), which were leafless plants of small stature and rhizomatous growth without a well-developed root system (Fig. 3c). In their larger axes only, both plants had xylem aligned in short radial files, but there is no evidence of rays, anticlinal cell division, or secondary phloem.

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So, in these instances, it seems likely that alignment of xylem cells took place through periclinal cell divisions during primary growth. The earliest evidence of secondary xylem in the fossil record also comes from euphyllophytes. Three occurrences have been reported recently from the Lower Devonian (Hoffman & Tomescu 2013), but the fossils are small and fragmentary, so much remains to be learned about their overall morphology. The earliest and most complete is the plant from Châteaupanne, Armorican Massif, France (late Pragian to earliest Emsian, c. 407 Ma), which is thought to closely resemble Psilophyton in overall morphology and mode of branching (Strullu-Derrien et al. 2010; Gerrienne et al. 2011; Strullu-Derrien et al., submitted). Anatomical resemblances extend to the overall shape of the xylem in transverse section (Fig. 4a), the development of the primary xylem (centrarch), and the P-type tracheids (Hartman & Banks 1980; Kenrick & Crane 1997a, b). The plant from Châteaupanne differs from Psilophyton in the presence of secondary xylem (Gerrienne et al. 2011; Strullu-Derrien et al., submitted). The xylem cells are aligned in radial files but unlike Psilophyton there is evidence of anticlinal cell division within the cell files (Fig. 4a; 5b, c), and possibly also the remains of a cambial layer (Gerrienne et al. 2011, fig. 4E). Rays are present (Fig. 4a), and they are probably uniseriate (Strullu-Derrien et al. submitted). The rays are rare and their form and size are very variable (Gerrienne et al. 2011, fig. 1A, F, G and fig. S2, S3 Supporting online material). Another distinctive feature, common in places, is the presence of tracheids with smaller radial diameter at the periphery of the wood. This configuration of cells might indicate the presence of a growth layer boundary within the wood or near its circumference or perhaps differences in the pattern of divisions of the fusiform initials along the cambial layer. Secondary xylem was also recently reported in an unnamed fragment of plant axis from New Brunswick, Canada (Late Emsian; c. 397 Ma). The plant had a protoxylem and a metaxylem that was oval-elongate in form (Fig. 4b). The secondary xylem was composed of P-type tracheids and contained relatively numerous rays (Fig. 4b and Gerrienne et al. 2011, fig. 1D). A third taxon, Franhueberia gerriennei (Gaspé, Canada; Late Emsian; c. 397 Ma) (Fig. 4c), is known from a short length of a rather distorted permineralized axis that also possessed secondary xylem of P-type tracheids. The rays are relatively numerous and they are regularly distributed (Hoffman & Tomescu 2013). Common features that can be observed or inferred in the three earliest woody plants known include 1) presence of a cambium with both tracheid and ray initials, 2) development of secondary growth in small axes, and 3) P-type tracheids. In all three cases absence of secondary phloem in the fossilized remains of the plants could reflect either true absence or inadequate preservation, so we cannot confidently define the cambium as either unifacial or bifacial. Although these fossils are fragmentary they provide evidence for a type of or approximation of secondary growth in small stature plants prior to the evolution of a distinctive leaf-stem-root organography (Hoffman & Tomescu 2013). Estimating the hydraulic properties of early fossil xylem

Our focus here is the hydraulic properties of early fossil xylem, which cannot be measured directly, but which can be estimated or modelled using approaches developed on

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living plants (Pittermann 2010). The hydraulic efficiency of xylem is proportional to the conduit diameter raised to the fourth power (Hagen-Poiseuille relation), so wide conduits are advantageous over narrow channels (Tyree & Zimmermann 2002). The consequences of this relationship on the early evolution of xylem was first clearly demonstrated by Niklas (1985), who documented an 18-fold increase in maximum tracheid diameter during the initial diversification of vascular plants in the Devonian Period. Here, the evolution of greater hydraulic efficiency was undoubtedly related to the great increases in plant size and complexity that characterized this period of time. Using an equation derived from the Hagen-Poiseuille relation, Cichan (1986) was the first to attempt to quantify specific conductance in the secondary xylem of fossil plants with the aim of comparing values obtained to those in modern groups. Tree forms and lianas were chosen from several major extinct groups of pteridophytes (Calamitaceae, Lepidodendraceae, Sphenophyllaceae) and gymnosperms (Cordaitaceae, Medullosaceae) of the Carboniferous Period. Results indicated that conductance in some of the most ancient woody groups was comparable to that in living plants. Highly effective conducting tissues had therefore developed relatively early in plant evolution. Also of interest is that some of the general relationships between wood anatomy, growth habit, and ecology known in living plants appeared to hold for these early fossils. Most Medullosans had an unusual and distinctive habit with slender stems that bore massive pinnately compound leaves. The vascular systems contained exceptionally wide tracheids, which is consistent with the inferred high evapotranspiration demands of the leaves (Wilson et al. 2008). The remarkably high inferred conductivities of the tracheids of Lyginopteris, Callistophyton, and especially Medullosa are similar to those of some vessel-bearing angiosperms, and the vascular anatomy indicates that they played little or no structural role in supporting stems. For some species this is suggestive of a semi-self-supporting vine-like or perhaps scandent habit (Wilson & Knoll 2010). It also indicates that these pteridosperms would not have fared well in seasonally arid or frost-prone environments, which is consistent with their inferred ecological setting as components of tropical floodplains floras (Wilson & Knoll 2010). The model used by Cichan (1986) treated the xylem in the stem as a single set of unobstructed pipes extending from roots to leaves and thus overestimated flow volumes through stems (Wilson et al. 2008). In other words, this simplification does not take into account the fact that tracheids have a finite length, typically in the range of about 0.5 mm to 4 mm in living conifers, and that flow between cells occurs through pits in the walls that offer significant additional resistance, reducing actual conductivity by well over 40% of that predicted by the Hagen-Poiseuille relation. Furthermore, the degree of this resistance varies with pit morphology, the porosity of the wall within the pit, and the size, number and distribution of pits, and values for this are not well characterized for living pteridophytes, many gymnosperms and their extinct relatives (Pittermann 2010). In general, we would expect the resistance of pits in the earliest fossils to be higher than those of modern plants due to the presence of an additional lignified perforate wall layer. Wilson et al (2008) took the estimation of hydraulic conductance in early vascular plants a step further by developing a model for water transport in xylem conduits that accommodated resistance to flow from pits and pit

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255.10--24 191 128 64 0

Figure 5. High-resolution Propagation Phase Contrast X-ray Synchrotron Microtomography (PPC-SRμCT) of 407 million year old fossil wood preserved in the mineral pyrite (FeS2) (Specimen CSD-07F-01, Université d’Angers, France). – a: Three-dimensional representation of part of pyritized axis of the plant from Châteaupanne, which has been virtually trimmed to a cubic volume. Xylem tracheids are visible in longitudinal, radial and transverse sections. – b: Transverse section extracted from the PPC-SRμCT part of xylem embedded in shale matrix; the mineral pyrite has been virtually dissected out leaving behind the organic framework of the tracheid cell walls. Scale bar = 250 μm. – c: Higher magnification of a transverse section extracted from the PPC-SRμCT part of xylem. At several places two xylem cell rows emanate from a single row (arrows). Scale bar = 50 μm. – d: Hagen-Poiseuille law prediction of lumens conductance based on a transverse section of part of xylem of the plant from Châteaupanne extracted from

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membranes, and they applied their model to compare hydraulic resistance in two of the extinct gymnosperms modelled by Cichan (1986) (Cordaitaceae, Medullosaceae) and living Pinus. One important distinction between the tracheids of these gymnosperms is that pit membranes in Pinus are differentiated into a torus and margo, whereas in Cordaitaceae and Medullosaceae the pit membranes are homogeneous. Also, the secondary xylem tracheids of extinct Medullosa are among the widest (commonly > 200 μm) and longest (>17.5 mm) known in seed plants (Andrews 1940). Results showed that medullosan tracheids had the capacity to transport water at flow rates more comparable to those of angiosperm vessels than to those characteristic of modern conifers and their ancient relatives (Cordaitaceae). Furthermore, results indicated that the tracheids operated at significant risk of embolism and implosion, making this plant unlikely to survive significant water stress. These observations are consistent with the general paleoecological interpretation of some medullosans as large-leaved lianas growing on tropical floodplains (DiMichele et al. 2006). This general approach was extended recently to make a broad comparison of hydraulic conductance across seed plants, based on a sample of 22 living and extinct species (Wilson & Knoll 2010). A morphometric approach was used comparing across groups the key factors governing hydraulic resistance: cell length, cell diameter, and pit resistance. Results showed that extinct coniferophytes fall within the range of living conifers. The efficiency of torus-margo pitting could be matched for species with homogeneous pit membranes by increasing pit area. Living cycads, extinct cycadeoids and Ginkgo overlapped with both conifers and vesselless angiosperms. However, three Palaeozoic seed plants (Lyginopteris, Callistophyton, Medullosa) stood out as occupying a unique portion of the morphospace. These extinct species therefore evolved a combination of tracheid morphologies and xylem architectures that lay outside the range observable in living gymnosperms and angiosperms. Much less is known about the comparative hydraulics of living ferns, horsetails, lycopods and their extinct relatives. Furthermore, in stem group vascular plants, tracheids differ in significant details of wall structure (e.g., S-type, G-type, P-type) to modern forms, further complicating the modelling of their hydraulic properties. Wilson and Fischer (2011) modelled the hydraulic resistance of the tracheid cell wall of the early fossil Asteroxylon using a simple scalariform pit model. However, the tracheids in Asteroxylon are of the G-type (Kenrick & Crane 1991); the pitting is not scalariform but basically annular developing into reticulate over part. Also, the model did not consider

←

PPC-SRμCT. In this section, the mineral pyrite has been virtually dissected out leaving behind the organic framework of the tracheid cell walls. The color intensity of each xylem conduit refers to its conductance performance ki. The individual conductivities ki of cells were computed using the hydraulic diameter approximation that was proposed by Sisavath et al. (2001) for undefined cross sections of conduits: 1 k i = —— μ DH i2 A i 32 where A i is the cross-sectional area of the conduit and DHi is its hydraulic diameter (StrulluDerrien et al., submitted).

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the effects of the presence of the additional perforate sheet of secondary wall material extending between annular bars, which significantly restricts the effective porosity of the tracheid wall. The flow from one cell to another through the wall of a G-type cell is effectively defined by the area of perforation in the secondary wall. The calculated values therefore underestimate the hydraulic resistance of this tracheid type. Despite difficulties such as these, the development of more sophisticated models incorporating pit architecture and distribution hold promise in furthering our understanding of the hydraulic properties of early wood. In addition to the theoretical considerations of modeling hydraulic conductance outlined above, there are technical and methodological issues concerning the characterization of both primary and secondary xylem in fossil plants. One set of issues relates to the imaging and measurement of tracheid cell walls. Typically, this is done by physical preparation of permineralized fossils to make transverse, radial and tangential sections (Jones & Rowe 1999). Measurements and imaging of tracheids would involve light microscopy. The method is destructive (i.e., results in loss of materials) and usually the number of preparations that one can make is rather limited. Scanning electron microscopy is used typically to develop detailed reconstructions of pit structure. Recently we showed how Synchotron microtomography can be used to document the structure of the earliest wood and to collect measurements to perform calculations on hydraulic conductivity (Fig. 5a–d and Strullu-Derrien et al. submitted). The potential of synchrotron microtomography

Synchotron microtomography (Feist et al. 2005; Lak et al. 2008; Tafforeau & Smith 2008) provides a new approach to investigating the structure and the hydraulic properties of early fossil wood (Strullu-Derrien et al., submitted). The method is (i) noninvasive and non-destructive, (ii) enables the visualization of wood volumes in three dimensions, and (iii) allows dynamic virtual dissection in any number of transverse, radial, and tangential sections to explore properties at the cellular level (Fig. 5a). For example, we were able to trace rays through a block of xylem and visualize in longitudinal tangential section the interface with xylem tracheids and rays. Adjacent tracheids have a double wall (consisting of the secondary walls of each tracheid whereas the interface with the ray shows only a single wall (tracheid wall) as cells within the ray are not preserved (Strullu-Derrien et al., submitted). Also virtual 3D histological sections (Fig. 5b, c) were generated from the reconstructed 3D volume, and used to estimate the hydraulic conductivity. Shape parameters (Ai and pi) of the cell lumens were measured in transverse section using the ImageJ software (Rasband 2012) (Fig 5d) (Strullu-Derrien et al., submitted). With standard paleobotanical methods, such observation and measurement require sectioning in three orientations (transverse, radial and tangential) and the using of etching agents, and they are destructive. Much early fossil wood is permineralized in pyrite (FeS2), which may become partially or completely oxidized (Kenrick 1999). The permineralization process results in mineralization that is believed to reflect the distribution of decay-resistant organics (i. e., lignin) within the original cellulose matrix

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of the cell wall (Kenrick & Crane 1991; Edwards 1993). Synchrotron microtomography makes possible the virtual removal of the mineral to reveal the coalified remains of the cell walls, thereby eliminating the need for chemical etching (Fig. 5b, c). Imaging is based on the attenuation of X-rays as they pass through the object, which is related to its density and chemical composition. It is possible that this approach would also work for woods preserved in other common minerals, including calcium, magnesium, and iron carbonates, and silicates. Tomography is dependent on density contrasts and it has to be noted that results that are very informative for fossils preserved in pyrite may be less so for fossils permineralized in calcite and silica. The method will probably be most effective where mineral replacement of the organic remains of the cell walls is incomplete. The volume of wood we reconstructed is small (Fig 5a), but in principle larger volumes can be imaged, allowing the accurate measurement of tracheid length and also an assessment of the distribution and density of pits within tracheid cell walls; however, there is going to be a trade-off between volume imaged and resolution. Larger volumes generally result in lower resolution. Our results show that the method works for early wood preserved in pyrite; it enables the dynamic 3D imaging of the wood, and it facilitates the analysis of its hydraulic and biomechanical properties in a non-invasive and non-destructive way. Conclusion

Understanding the early evolution of hydraulic conductivity in wood requires a detailed documentation of early fossils. The xylem in fossils is often well-preserved providing information on the structure of the tracheids and the general form and composition of the vascular system. Recent studies show that early fossil woods differ in many key respects to those of modern gymnosperms (e.g., tracheid size, secondary wall thickenings, lignin chemistry, cambium development). Hydraulic conductivity cannot be measured directly in fossils, but estimates can be obtained by measuring various properties of the fossilized water-conducting cells and using these as values in increasingly sophisticated biophysical models. This approach holds great promise in furthering our understanding of the hydraulic properties of early wood however modelling pit resistance in early fossils remains challenging. Synchrotron microtomography is a flexible new and non-invasive tool for the study of permineralized woods that enables their dynamic virtual dissection. The method is effective for woods preserved in pyrite (FeS2 ), and might also prove effective for woods preserved in other common minerals. The method facilitates the collection of measurements needed to calculate hydraulic conductivity, in a non-destructive way and for very small samples, which is a clear advantage. The fossil record shows that wood evolved in small stature plants prior to the evolution of a distinctive leaf-stem-root organography. It also demonstrates that the suite of characteristics that comprise wood in modern gymnosperms assembled over a period of time. Results are beginning to show combinations of features in fossil woods that are outside of the range observed in modern plants. Knowledge of the hydraulic and the biomechanical properties of fossils woods can also help inform on the growth habit and ecology of extinct plants.

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The authors thank Pieter Baas and Elisabeth Wheeler, Editors-in-Chief of the IAWA Journal for their invitation to write this article. They thank Dianne Edwards, Patricia G. Gensel (who studied the plants from New Brunswick) and Alexandru M. Tomescu for permission to reproduce figures. C.S-D received financial support from the European Commission under the Marie Curie Intra-European Fellowship Programme FP7-People-2011-Symbionts.

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Taylor TN, Taylor EL & Krings M. 2009. Paleobotany: the biology and evolution of fossil plants. Ed. 2. Elsevier, Academic Press, New York. Tyree MT & Zimmermann MH. 2002. Xylem structure and the ascent of sap. Springer, Berlin. Wang DM, Hao SG & Wang Q. 2003. Tracheid ultrastructure of Hsua deflexa from the Lower Devonian Xujiachong Formation of Yunnan, China. Int. J. Plant Sci. 164: 415–427. Wilson JP & Fischer WW. 2011. Hydraulics of Asteroxylon mackei [sic], an early Devonian vascular plant, and the early evolution of water transport tissue in terrestrial plants. Geobiol. 9: 121–130. Wilson JP & Knoll AH. 2010. A physiologically explicit morphospace for tracheid-based water transport in modern and extinct seed plants. Paleobiol. 36: 335–355. Wilson JP, Knoll AH, Holbrook NM & Marshall CR. 2008. Modeling fluid flow in Medullosa, an anatomically unusual Carboniferous seed plant. Paleobiol. 34: 472–493. Accepted: 8 September 2013

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Axial conduit widening in woody species: a still neglected anatomical pattern Tommaso Anfodillo*, Giai Petit and Alan Crivellaro Università degli Studi di Padova, Dipartimento Territorio e Sistemi Agro Forestali, Viale dell’Università 16, 35020 Legnaro (PD), Italy *Corresponding author; e-mail: [email protected]

Abstract

Within a tree the lumen of the xylem conduits varies widely (by at least 1 order of magnitude). Transversally in the stem conduits are smaller close to the pith and larger in the outermost rings. Axially (i.e. from petioles to roots) conduits widen from the stem apex downwards in the same tree ring. This axial variation is proposed as being the most efficient anatomical adjustment for stabilizing hydraulic path-length resistance with the progressive growth in height. The hydrodynamic (i.e. physical) constraint shapes the whole xylem conduits column in a very similar way in different species and environments. Our aim is to provide experimental evidence that the axial conduit widening is an ineluctable feature of the vascular system in plants. If evolution has favoured efficient distribution networks (i.e. total resistance is tree-size independent) the axial conduit widening can be predicted downwards along the stem. Indeed, in order to compensate for the increase in path length with growth in height the conduit size should scale as a power function of tree height with an exponent higher than 0.2. Similarly, this approach could be applied in branches and roots but due to the different lengths of the path roots-leaves the patterns of axial variations of conduit size might slightly deviate from the general widening trend. Finally, we emphasize the importance of sampling standardization with respect to tree height for correctly comparing the anatomical characteristics of different individuals. Keywords: Tree height, hydraulic resistance, xylem, evolution. Introduction

The identification and classification of wood anatomical traits are of fundamental interest to wood anatomists, botanists and plant ecologists. Indeed, variations of such traits in different species and environments largely determine the mechanical and technological properties of wood and thus its economic value. Wood anatomists have therefore established a very detailed set of anatomical traits for describing the astonishing variety of xylem anatomical features that have been evolved by terrestrial plants (Wheeler et al. 1989). These anatomical variations form a basis to hypothesize adaptive strategies as drivers of much of the wood anatomical diversity that has resulted through evolution (Carlquist 1975; Baas et al. 2004). © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000030

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Furthermore, some anatomical traits were used as predictors of the capacity of a given plant to survive in specific environmental conditions. For example, the diameter of the conduits in the xylem is believed to be of major adaptive importance (Sperry et al. 2006). In a seminal manuscript (actually the most cited paper ever published in IAWA Journal) (Tyree et al. 1994) it is stated that a general trend in xylem conduit diameter can be derived from several anatomical observations (Carlquist 1975): “wet-warm environments tend to favour species with wide conduits whereas cold or dry environments tend to favour species with narrow conduits.” A more recent meta-analysis (Wheeler et al. 2007) further supported the idea that few wide vessels are associated with tropical environments and many narrow vessels are associated with high latitudes and environments with prolonged periods of low water availability. Interestingly they also noted that vessel diameter is strongly related to habit (i.e. height). Shrub species have the highest proportion of narrow vessels (< 50 µm) whereas wide vessels (> 200 µm) are virtually absent. In trees, on the contrary, wide vessels are very common. Since the diameter of xylem conduits seems to be a crucial parameter in plant physiological ecology, it is very important to identify why and how conduits size changes within a plant, in plants of different height and in different growing conditions. Therefore, a wood anatomist /ecologist aiming to study the variation of conduit diameter should ask him-/herself the following questions: where should I localize the sampling point? Does conduit diameter vary in the different parts of plants (roots, branches, stem)? If yes, how large is the variation? How can I standardize the measurements? We suspect that many readers (if not all) might comment that these questions have already been largely clarified (see for example Tyree & Zimmermann 2002). Notable examples can be found in Sanio (1872) who described “Sanio’s trends” in an individual of Pinus sylvestris. In that paper the author reported an exhaustive (although not always perfectly clear) description of the trends in tracheids diameter along the stem, branches and roots: in the stem conduits widen basipetally (i.e., increase in diameter axially towards the roots), whereas in roots they are generally wider than in the stem. His observations were very useful and have been substantially confirmed by successive measurements (Zimmermann 1978; Tyree & Zimmermann 2002). In addition, the latter authors found that the axial variation (along the stem) is far from being constant (i.e., linear with the axis length): conduit diameter largely changes near the treetop and then the rate of diameter variation progressively declines becoming rather constant further down at the stem base. This pattern seems to be particularly useful in terms of both hydraulic safety and efficiency, because on the one hand it provides the distal regions of the xylem pathways (where tensions are higher) with the conduits most resistant to cavitation (Hacke et al. 2001), on the other it confines the greater part of total hydraulic resistance towards the downstream ends of the flow path (i.e. the leaves) (Becker et al. 2000; Petit & Anfodillo 2009). A further and decisive improvement in understanding the structure of the xylem vascular system (and its basic physical requirements) was provided by West et al. (1999) with the so-called “WBE model”: they were the first to propose an explanation for why and how much the root-to-leaves column of vascular conduits should vary in width

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axially, as previously observed. The theoretical approach of West et al. (1999) played a pivotal role in understanding axial variation of the pipelines’ width. Comments on the model structure, allometric consequences and also the relevant flaws can be easily found elsewhere (Mencuccini 2002; Kozlowski & Konarzewski 2004; McCulloh & Sperry 2005; Coomes 2006; Etienne et al. 2006; Petit & Anfodillo 2009). The WBE model simply proposes a plant formed by a bundle of tubes of the same length running in parallel from the roots to the leaves. Notably the tubes are not cylindrical (as in previous hydraulic models) but are tapered (i.e. they widen towards the stem base). We focus our attention on the straightforward and revealing structure of the WBE model because, differently from all other previously-cited anatomical studies, it allows us to predict the variation of conduit diameters along the stem axis or in different individuals throughout ontogenesis. Our aim is to present the “anatomical structure” of the plant modelled by WBE in detail and to prove that such a predictable pattern is very close to the one observable in nature. We are also convinced that the awareness of the ubiquity of this anatomical feature could help wood anatomists to standardize and functionally compare their measurements. We are naturally aware that the tree modelled by WBE can only represent an “idealized plant” and that some oversimplifications might be problematic (for example, the assumption that the “tubes” are all of the same length). Our intention is to keep the most valuable idea of the WBE model (and it is really useful) in the knowledge that a lot of work still needs to be done for modelling the hydraulic architecture of trees in detail. Similarly, it is clear that a simple little paper airplane differs enormously from a Boeing 737 but, notably, it does have the same essential property: it flies! Why should conduit diameter vary with plant height?

Anatomy and physiology are two sides of the same coin. If a general plant requirement must be guaranteed (e.g., maintaining leaf efficiency during different life stages) then the anatomy of the plant should be adjusted accordingly. Useful information about anatomical changes can therefore be drawn from models aimed at describing how trees work. A brief “foray” into the WBE model is needed to explain why conduit diameter should vary with plant height. In short, the idealized plant modelled by the WBE model is very similar to that proposed by Shinozaki et al. (1964): the tree can be seen as a “set of bundles” running in parallel from roots to leaves. These bundles (i.e. axial chains of xylem conduits) are connected to a fixed set of leaves (one-to-one in the simplest case). Notably these bundles are believed to be all of the same length: this simplification, which is evidently not true in real plants, will be discussed later. Overall, the model predicts a complete independence among the different pathways. This condition might be in agreement with the idea of “plant segmentation” (Tyree & Zimmermann 2002) and would bring a selective advantage in ensuring both the

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Figure 1. Variation of the earlywood tracheid diameter along the stem axis in the same annual ring (Picea abies (L.) Karst.): this variation is referred to as “conduit widening” (direction towards the stem base) or “conduit tapering” if the direction of the water flow is considered. All the sections have the same degree of magnification (indicated at the bottom of the figure: bar = 50 μm).

optimum supply to parts of the plant that are, at the same time, subjected to different metabolic rates (e. g. sun or shade leaves) and for better confining system failures (e.g. embolisms, pathogens). In order to maintain the leaf efficiency as constant as possible during ontogeny (i.e., when the tree grows) a new “ingredient” must be introduced to the simple “pipe model”: the pipes are not cylindrical but they have a different diameter along the longitudinal axis (i.e. they widen basipetally) (Fig. 1). The variation of conduit diameter downwards in the stem (conduit widening, also known as conduit tapering) is predicted to be a power law according to which the variation of conduit diameter (Dh) with the distance from the tree top (L) (note that it is the inverse of tree height) will be: Dh ∝ L b,

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where b is the exponent of the power function, which accounts for the relative variation. In the case of b = 0 there is no axial variation and the shape of the tube is cylindrical (like the original version of the pipe model). The rate of increase in hydraulic resistance with tree height strongly depends on how conduits vary in size along the stem. When xylem cells increase in diameter from the stem apex to the base a higher proportion of resistance is confined towards the apex. The higher the degree of widening, the greater is the magnitude of resistance confined to the apex. This implies that with further stem elongation, widening conduits towards the stem base would allow for a compensatory effect on the path length resistance, the efficiency of which is higher for higher degrees of widening (Becker et al. 2000; Petit & Anfodillo 2009) (Fig. 2). Notably, the degree of widening predicted by the WBE model (b = 0.25) can be considered as a threshold value above which the independence of the hydraulic resistance from the total path length (i.e. tree height) is guaranteed. The WBE theory predicts that evolution has acted in such a way that the degree of widening is the minimum required to make the resistance independent of the tree height (Enquist 2003). If true, then the leaf metabolic efficiency will be kept “invariant” through ontogeny, i.e., water supply to the leaves will be similar in both small and very tall trees. Figure 2. The effect of progressive degree of widening (or tapering) of vascular conduits (i.e. the 150 scaling exponent b in determining the total hydraulic resistance, relative to the resistance of the most apical conduit ( RTOT / RAPEX ), 100 with the increasing length (L) of a single pipeline. Theory assumes that plants should ap50 proach the minimum degree of widening (b = 0.2) required to fully compensate for the pro 0 5 10 15 20 25 30 gressive increase in hydraulic re L (m) sistance (on the y-axis) with the growth in height (i. e. path length on the x-axis). The scaling of the total resistance at different exponent b can be predicted on the basis of physical laws. b=0 b = 0.1 b = 0.15 b = 0.2 b = 0.3

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In order to compare real trees with the “idealized plant” of the WBE model it is necessary to take into account the approximation of the WBE model in estimating the tree height. Doing so the minimum scaling exponent b, relative to the scaling of the conduit dimension versus the distance from the treetop, assumes the value of about 0.20 (see details in Anfodillo et al. 2006). This value (exponent similar to or higher than 0.20) means that in a real tree (not in the idealized plant of the WBE model) the vascular network is structured to compensate for the increase in plant height. Exponents in agreement with the predicted one have been repeatedly measured in plants of different sizes and environments (Anfodillo et al. 2006; Coomes et al. 2007; Petit et al. 2008, 2009; Lintunen & Kalliokoski 2010; Petit et al. 2010, 2011; Bettiati et al. 2012; Olson & Rosell 2012) (Fig. 3).

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Acer pseudoplatanus Eucalyptus regnans Fraxinus excelsior Nothofagus solandri Larix decidua Picea abies Pinus cembra Pinus sylvestris

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Figure 3. A meta-analysis of the axial variation of xylem conduit diameter (Dh) with the distance from the stem apex (L) of different species (inset). Data refer to measurements in stems and branches. Literature data are: Acer pseudoplatanus from Petit et al. (2008); Eucalyptus regnans from Petit et al. (2010); Fraxinus excelsior from Anfodillo et al. (2006) and Bettiati et al. (2012); Nothofagus solandri from Coomes et al. (2007); Larix decidua from Anfodillo et al. (2006) and Petit et al. (2009); Picea abies from Anfodillo et al. (2006), Coomes et al. (2007), Petit et al. (2009) and Petit et al. (2011); Pinus cembra from Petit et al. (2009); Pinus sylvestris from Coomes et al. (2007) and original data. The parallel grey lines indicate the power scaling with exponent b = 0.20. Lines change in relation to different intercepts (i.e. conduit size at the plant apex).

Figure 4. Variation of conduit diameter at the stem base (in 4 classes: < 20; 20–50; 50–100; 100–200 μm) in relation to averaged class of plant height (4 classes). Data from Fritz Schweingruber relative to 3339 different angiosperm species mainly from the northern hemisphere (available at: www.wsl.ch/dendro/xylemdb/index. php). The tallest plants have formed the widest conduits.

Averaged conduit lumen diameter (µm)

With the knowledge gained so far it is logical to expect that the relatively largest conduit dimensions at the stem base should be found in tall trees and, in contrast, small plants will have relatively small conduits: this general pattern in conduit size is therefore the consequence of the plant size. A meta-analysis on available data shows that conduits at the stem base are generally wider in taller plants (Fig. 4 and see also Wheeler et al. 2007) 200

Fritz Schweingruber dataset 3339 different angiosperms

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21 64 188 468 Averaged class plant height (cm)

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Solving the chicken-egg dilemma

As mentioned above, Tyree et al. (1994) wrote that small conduits were generally measured in plants of dry/cold environments and therefore it could be speculated that, in a low-resources site, natural selection acts against species with relatively large conduits. However, the previous considerations showed that conduit size is closely correlated to tree height. Now, since tree height is negatively affected in low-resources sites so plants are generally small, it is obvious to expect narrower conduits. This circular reasoning could be solved by disentangling the role of tree size in determining the conduit size from that of resources’ availability. In a recent elegant analysis Olson and Rosell (2012) compared the conduit dimension at the stem base, in 142 different species growing in 5 sites within a huge gradient of water availability (annual precipitation from about 800 to 3500 mm). They then plotted the conduit dimension of each plant versus its stem diameter (which is allometrically linked to tree height) in each site. In this way they were able to compare the intercept of each regression line, i.e. the average conduit dimension in plants with the same basal diameter. They clearly demonstrated that the conduit size in plants growing in the wettest and driest sites did not differ. The conclusion is that plants of the drier sites are generally smaller and therefore have narrower conduits but, at the same size their conduits substantially do not differ from those of the species growing in the wetter sites. This places full emphasis on how important it is to standardize the collection of samples in relation to plant height if the aim is to evaluate the adaptive consequences of given anatomical structures. The awareness of the ubiquity of the axial widening pattern could therefore help in correctly interpreting the anatomical traits. Even more importantly, results indicate that conduit diameter is strictly related to tree height but little or not at all to the age of the plant. We believe that it is time to revise the common belief that this wood trait is substantially age-dependent. What occurs in the cambial zone and the cascade of events leading to a final conduit dimension are primarily related to the distance of the cambial cells from the apex (and, obviously, to environmental constraints). Indeed, it was demonstrated that the number of days in which the forming cells remain in the expansion phase is directly related to distance from the tree top (L) (Anfodillo et al. 2012). Cambial activity and the cell formation is basically height- and not age-related, as confirmed by Petit et al. (2008), who showed that apical shoots from very tall parent Acer pseudoplatanus trees, grafted onto 1-year-old rootstocks, developed vessels of similar sizes to those of young trees of similar height. One of the important conclusions of this paper is that, when dealing with variation of wood traits (e.g. density, cell dimension, fraction of latewood etc.) within a functionalbased approach, it would be better to change the traditional idea of “age-dependency” with the more correct concept of “size-dependency”. Pervasiveness of the pattern

One of the most unrealistic assumptions of the WBE model is that it considers an idealized plant formed by “pipes” of the same length. However, in trees branches have different length: some of them are very close to the ground and others near the treetop

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Figure 5. Right: Variation of conduit diameter (Dh) at the apex of the branches in relation to the length of the path PL (calculated from root to apex). Left: example of two paths (grey thick lines) with different length in a stylized Fraxinus excelsior (number of internodes are also indicated). The diameter of apical conduits increases with the increase of the path: this anatomical feature should compensate for the different length of the paths. Shorter paths have smaller apical conduits thus the total hydraulic resistance can be maintained very similarly among different paths (data from Bettiati et al. 2012).

(Fig. 5). Thus a basic assumption of the model appeared to be violated in nature. How can a plant cope with the variable length of the paths? The simple underlying idea is that a plant should adjust the structure (i.e. anatomy) of the vascular conduits to achieve a condition of equiresistance of all roots-to-leaves paths. If this condition was not satisfied then water would flow mainly throughout the paths with lower hydraulic resistance. But this would be detrimental to achieving similar water supply to all leaves. There is not much information about the hydraulic architecture of the whole tree carried out with appropriate sampling (i.e. taking into account the distance of the samples from the tree top). Some examples can be derived from the book by Tyree & Zimmermann (2002) but in many cases the exact sampling distance is not specified thus making interpretation of results difficult. Recent anatomical analyses (Bettiati et al. 2012) showed that diameter of apical conduits in branches (collected just below the apex) are significantly different, with the widest diameters found in the longest root-toleaves paths (Fig. 5). For example, in very short branches in the basal part of the crown (short path) apical conduits are relatively small (2–3 times smaller than the conduits in the longest paths/branches). This seems a very simple and coherent adjustment in order to guarantee similar resistance in all paths and therefore equal water delivery to the different parts of the crown. Generally, it clearly emerges that the anatomical feature of one part of a plant (e.g., an apex of a certain branch) is strictly linked to the anatomy of the whole individual. Thus in spite of the near independence of all different paths they are anatomically structured in order to supply all the leaves in the different parts of the crown at similar rates: this result is equivalent to that obtained considering all the tubes of the same length (as in the simple WBE idealized plant).

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Figure 6. Variation of conduit diameter (Dh) with the distance from the apex in two different branches of a 9.5 m tall poplar tree (Populus × canadensis). The exponent b slightly changes (the 95 % CI is also indicated). In less than 2.0 m in length the conduit diameter changes by a factor of 2.5 (from about 20 to 50 μm).

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Since the “tubes” run from roots to leaves, the same general pattern of conduit widening should in principle be preserved along the longitudinal axes of both branches and roots. Systematic measurements of conduit widening in branches are even more difficult to find than the axial variation of conduit size in stems. However, measurements of hydraulic permeability (a proxy for conduit diameter) in branches generally showed an increase with branch diameter (i.e. towards the stem) (Tyree & Alexander 1993; Jerez et al. 2004; Sellin & Kupper 2007) thus demonstrating that conduits become wider towards the branch base. An analysis on the axial variation of conduit diameter in branches of a poplar tree showed a common pattern of widening compatible with the value of 0.20 but short branches also showed a lower degree of tapering (0.16– 0.14) (Fig. 6). Similarly to stem anatomy the variation of conduit dimension along the branch strictly depends on the distance from the branch tip. The conduit diameter might easily vary by a factor of about 2 for a variation of 1 m in position of the sample towards the stem. It is evident how important it is to also consider the pattern of conduit widening in branches. Any measurement, for example, of hydraulic conductivity, which is taken on small pieces of branches, must be normalized for the distance from the branch tip. Otherwise the results might be dependent only on the position of the sample and thus become meaningless.

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Xylem conduits are believed to continue to increase in diameter downwards also in roots. Indeed, in general, vessel diameter and length in woody roots exceed those in stems of comparable diameter (Tyree & Zimmermann 2002), thus the widest cells of the whole plant are found in roots. This pattern was observed in shrubs of cold deserts where the mean vessel diameter was about 2 times wider than in the stem (Kolb & Sperry 1999), in nine Mediterranean woody species (Martinez-Vilalta et al. 2002) where the diameter of conduits was always wider in roots than in stems, and in conifers (Petit et al. 2009). However, in very short superficial roots it is not uncommon to find relatively narrow cells (even narrower than in the stem). This is probably due to the fact that in roots (as occurs in branches) the length of the paths might differ significantly so the plant would compensate for possible differing hydraulic resistance by adjusting the conduit dimension accordingly (i.e. narrower conduits in shorter roots). Notably in the roots the allometric relationships of root diameter versus distance from the tree top has an opposite sign compared to the stem (in roots the exponent is negative) showing that the mechanical constraints in roots differ from those of the stem and branches. Nonetheless the hydraulic constraints in roots are similar to the other organs so the degree of widening is supposed to be similar in all parts of the plants. Measurements in roots indicate that conduit widening seems to be a stable property of the whole xylem architecture, with the widest conduits very close to the root tips (as predictable from the hydraulic requirements) (Petit et al. 2009). However, further measurements are needed to clarify the pattern of conduit size in roots because they are characterized by a branching network with huge variations in the length of the different conductive paths. A still unanswered question

One of the most intriguing physiological questions related to axial widening is: how can a plant so precisely regulate the axial conduit dimensions along a path that may exceed 150 m in length (stem and roots in the tallest trees)? Wood formation in trees is a dynamic process, strongly affected by environmental conditions, including nutrient availability and climatic changes (Oribe et al. 2003; Arend & Fromm 2007; Sorce et al. 2013; Prislan et al. 2013). Despite the relevant role of cambium tissues in plants, few studies have dealt with the molecular and structural mecanisms at the basis of its functionality (Deslauriers et al. 2009; Berta et al. 2010). According to a recent study, wider cells along the stem are those staying longer in the distension phase during xylogenesis (Anfodillo et al. 2012). Therefore, it is likely that plants adopt a mechanism to modulate the time for cell enlargement to precisely design xylem conduits optimally widened from the stem apex downwards for hydraulic purposes. A candidate hypothesis proposes the polar transportation of phyto-hormones, particularly auxin (IAA), as the mechanism of control of the dimension of xylem cells along the stem (Aloni 2001; Aloni et al. 2003). The IAA is produced mainly in the developing buds and shoots (Uggla et al. 1998; Scarpella & Meijer 2004) and is transported basipetally along the cambial zone (Sundberg et al. 2000) from leaves to

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roots (Aloni 2001; Muday & DeLong 2001). Moreover, the IAA concentration generally decreases from the stem apex to the base (Lovisolo et al. 2002). According to the six point hypothesis (Aloni & Zimmermann 1983), high concentrations of IAA would accelerate the cellular differentiation, thus reducing the time period for the cellular distension phase. This new research topic should be promoted given the pivotal role in regulating the efficiency of water transport. We hope that this simple manuscript might encourage some scientists to clarify the physiological mechanisms related to axial conduit widening. Conclusions

The xylem conduit size in stems, branches and roots appeared invariably dependent on the distance from the top: moving downwards (basipetally) the conduits are gradually wider and this is a necessary anatomical feature for stabilizing the hydrodynamic resistance with tree height. Thus the requirement for maintaining the efficiency of water transport throughout the plant ontogenesis in all crown parts is achieved by shaping a widened anatomical structure. We believe that the axial conduit widening can no longer be neglected because it offers a clear and universal physiological explanation of the anatomical changes carefully observed by Sanio more than a century ago. Acknowledgements The manuscript was funded by the University of Padova, project UNIFORALL (CPDA110234). It was also inspired and supported by the EU COST Action FP1106. Alan Crivellaro was supported by the University of Padova (Assegno di Ricerca Junior CPDR124554 /12). The authors thank A. Garside for checking the English text.

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Petit G & Anfodillo T. 2009. Plant physiology in theory and practice: An analysis of the WBE model for vascular plants. J. Theor. Biol. 259: 1–4. Petit G, Anfodillo T, Carraro V, Grani F & Carrer M. 2011. Hydraulic constraints limit height growth in trees at high altitude. New Phytol. 189: 241–252. Petit G, Anfodillo T & De Zan C. 2009. Degree of tapering of xylem conduits in stems and roots of small Pinus cembra and Larix decidua trees. Botany 87: 501–508. Petit G, Anfodillo T & Mencuccini M. 2008. Tapering of xylem conduits and hydraulic limitations in sycamore (Acer pseudoplatanus) trees. New Phytol. 177: 653–664. Petit G, Pfautsch S, Anfodillo T & Adams MA. 2010. The challenge of tree height in Eucalyptus regnans: when xylem tapering overcomes hydraulic resistance. New Phytol. 187: 1146–1153. Prislan P, Čufar K, Koch G, Schmitt U & Gričar J. 2013. Review of cellular and subcellular changes in cambium. IAWA J. 34: 391– 407. Sanio K. 1872. Über die Größe der Holzzellen bei der gemeinen Kiefer (Pinus sylvestris). J. Wiss. Bot. 8: 401– 420. Scarpella E & Meijer AH. 2004. Pattern formation in the vascular system of monocot and dicot plant species. New Phytol. 164: 209–242. Sellin A & Kupper P. 2007. Effects of enhanced hydraulic supply for foliage on stomatal responses in little-leaf linden (Tilia cordata Mill.). Eur. J. Forest Res. 126: 241–251. Shinozaki K, Yoda K, Hozumi K & Kira T. 1964. A quantitative analysis of plant form - The pipe model theory I. Basic analyses. Jap. J. Ecol. 14: 94–105. Sorce C, Giovannelli A, Sebastiani L & Anfodillo T. 2013. Hormonal signals involved in the regulation of cambial activity, xylogenesis and vessel patterning in trees. Plant Cell Rep. 32: 885–898. Sperry JS, Hacke UG & Pittermann J. 2006. Size and function in conifer tracheids and angiosperm vessels. Amer. J. Bot. 93: 1490–1500. Sundberg B, Uggla C & Tuominen H. 2000. Cambial growth and auxin gradients. In: Savidge RA, Barnett JR & Napier R (eds.), Cell and molecular biology of wood formation: 169–188. BIOS Scientific Publishers Ltd., Oxford. Tyree MT & Alexander J. 1993. Hydraulic conductivity of branch junctions in three temperate tree species. Trees 7: 156–159. Tyree MT, Davis SD & Cochard H. 1994. Biophysical perspectives of xylem evolution: is there a tradeoff of hydraulic efficiency for vulnerability to dysfunction? IAWA J. 15: 335–360. Tyree MT & Zimmermann MH. 2002. Xylem structure and the ascent of sap. Springer, Berlin. Uggla C, Mellerowicz EJ & Sundberg B. 1998. Indole-3-acetic acid controls cambial growth in Scots pine by positional signaling. Plant Physiol. 117: 113–121. West GB, Brown JH & Enquist BJ. 1999. A general model for the structure and allometry of plant vascular systems. Nature 400: 664–667. Wheeler EA, Baas P & Gasson PE (eds.). 1989. IAWA List of microscopic features for hardwood identification. IAWA Bull. n. s. 10: 219–332. Wheeler EA, Baas P & Rodgers S. 2007. Variations in dicot wood anatomy: a global analysis based on the InsideWood database e.a. IAWA J. 28: 229–258. Zimmermann MH. 1978. Hydraulic architecture of some diffuse-porous trees. Can. J. Bot. 56: 2286–2295. Accepted: 29 August 2013

Rosner IAWA – Hydraulic Journaland 34 biomechanical (4), 2013: 365–390 optimization

HYDRAULIC AND BIOMECHANICAL OPTIMIZATION IN NORWAY SPRUCE TRUNKWOOD – A REVIEW Sabine Rosner Institute of Botany, BOKU Vienna, Gregor Mendel Str. 33, 1180 -Vienna, Austria E-mail: [email protected]

abstract

Secondary xylem (wood) fulfills many of the functions required for tree survival, such as transport of water and nutrients, storage of water and assimilates, and mechanical support. The evolutionary process has optimized tree structure to maximize survival of the species, but has not necessarily optimized the wood properties needed for lumber. Under the impact of global warming, knowledge about structure-function relationships in tree trunks will become more and more important in order to prognosticate survival prospects of a species, individuals or provenances. Increasing our knowledge on functional wood anatomy can also provide valuable input for the development of reliable, fast, and at best quasi-non-destructive (e.g. wood coring of mature trunks) indirect screening techniques for drought susceptibility of woody species. This review gives an interdisciplinary update of our present knowledge on hydraulic and biomechanical determinants of wood structure within and among trunks of Norway spruce (Picea abies (L.) Karst.), which is one of Europe’s economically most important forest tree species. It summarizes what we know so far on 1) withinring variability of hydraulic and mechanical properties, 2) structure-function relationships in mature wood, 3) mechanical and hydraulic demands and their tradeoffs along tree trunks, and 4) the quite complex wood structure of the young trunk associated with mechanical demands of a small tree. Due to its interdisciplinary nature this review is addressed to physiologists, foresters, tree breeders and wood technologists. Keywords: Biomechanics, functional anatomy, hydraulic vulnerability, Norway spruce, Picea abies, tracheid, water transport, wood density. INTRODUCTION

Global change is expected to increase the frequency of extreme weather and climate events such as heat waves, drought periods and storms (Schär et al. 2004; Salinger 2005; IPCC 2012; Semenov 2012). As a consequence, plant productivity and growth will be affected in many regions (Ciais et al. 2005; Bréda et al. 2006; Reich & Oleksyn 2008; Kullman & Öberg 2009; Choat et al. 2012; Williams et al. 2012). Continued refinement and extension of models linking between plant hydraulics, mechanics and ecosystem functioning will help to gain information on broader patterns in productivity that are © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000031

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related to plant water use (Geßler et al. 2007; McDowell et al. 2008; Sperry et al. 2008; Meinzer et al. 2009, 2010; Barnard et al. 2011). This knowledge can be applied to prognosticate survival prospects of woody species (Pockman & Sperry 2000; Maherali et al. 2004; Hukin et al. 2005; Dalla-Salda et al. 2009, 2011; Choat et al. 2012), or to screen for less drought-sensitive clones (Rozenberg et al. 2002; Monclus et al. 2005; Cochard et al. 2007; Rosner et al. 2007, 2008), or provenances (Peuke et al. 2002; Kapeller et al. 2012). Screening for hydraulic and mechanical performance demands, however, easily applicable methods. Increasing our knowledge on structure-function relationships in trunkwood can provide valuable input for the search on reliable, fast, and at best quasi-non-destructive (e.g. wood coring of mature trunks) methodologies for indirect assessment of wood biological functions. This review gives an overview on our present knowledge on structure-function relationships in the trunkwood of Norway spruce (Picea abies (L.) Karst.), which is one of central and northern Europe’s economically most important forest tree species (Bergh et al. 2005). Norway spruce can be found up to the alpine timberline (Mayr et al. 2002, 2003) as well as in the far north (Tollefsrud et al. 2008). Problems associated with weak adaptation of Norway spruce to environmental conditions in lowland regions, such as top dieback, damage due to wind and snow loads and subsequent bark beetle mass outbreaks which are favoured by drought stress of the host trees frequently cause enormous economic losses (Solberg 2004; Andreassen et al. 2006; Schlyter et al. 2006). Interdisciplinary research on structure-function relationships within conifer trunks started in the late 90-ties when Mencuccini et al. (1997) published their work on biomechanical and hydraulic determinants of tree structure in Scots pine (Pinus sylvestris L.). Experimental studies on structure-function relationships within conifer trunks are, however, still quite scarce and much more literature exists on pine species than on spruce species (Lachenbruch et al. 2011). The most entirely investigated conifer species are Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) (e.g. Domec & Gartner 2001; Spicer & Gartner 2001; Domec & Gartner 2002a, 2002b; Dunham et al. 2007; Domec et al. 2009) and Ponderosa pine (Pinus ponderosa Dougl. ex Laws.) (e.g. Bouffier et al. 2003; Domec & Gartner 2003; Domec et al. 2005; Domec et al. 2009; Barnard et al. 2011). Jagels et al. (2003) and Jagels and Visscher (2006) provided us also insights into the quite unique structure-function relationships within the main trunk of Metasequoia (M. glyptostroboides Hu et Cheng). Mayr et al. (2002) were the first to relate hydraulic performance in Norway spruce leader shoots to wood structure and discussed the results upon mechanical demands before Rosner and colleagues published their work series on biomechanical and hydraulic optimization within and among Norway spruce trunks (Rosner et al. 2006, 2007, 2008, 2009, 2010; Rosner & Karlsson 2011; Rosner et al. 2012). The dataset developed during the past years on structure-function relationships in Norway spruce trunkwood is quite unique; Norway spruce was used e.g. as a model species to develop new analysis methods to assess hydraulic vulnerability and interdisciplinary research combining wood anatomy, wood physiology and wood technology was helpful to get a broader view of structure-function relationships within the tree trunk. The aim of this review is to give an update of our present knowledge on hydraulic

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and biomechanical determinants of wood structure within and among Norway spruce trunks and is addressed to physiologists, foresters, tree breeders as well as to wood technologists. The tracheid: a multitasking wood element Tracheids fulfill many of the functions required for tree survival, such as water transport, water storage and mechanical support. Norway spruce secondary xylem (wood) consists to more than 90% of tracheids (Fig. 1), the remaining tissue is parenchyma (Brändström 2001). Cell wall layers of tracheids consist of 40–50 % cellulose, 25–30 % lignin and 20–25% hemicellulose (Plomion et al. 2001). Cellulose chains are arranged in microfibrils, which are unordered in the primary wall, but highly ordered in the thickest layer of the secondary cell wall (S2 ) (Booker & Sell 1998). Reviews on ultra-structural features of the different cell wall layers with special reference to Norway spruce tracheids can be found elsewhere (Neagu et al. 2006; Jungnikl et al. 2008; Salmén & Burgert 2009). Each tracheid is connected with its adjacent tracheids by bordered pits (Fig. 2) consisting of a porous membrane held in a pit chamber. Pit membranes in Norway spruce are of the torus-margo type, owing a thin and porous margo with a thickened torus (Liese & Bauch 1967; Greaves 1973; Gregory & Petty 1973). According to its hydraulic and biomechanical tasks within the trunk, the morphology of a tracheid varies considerably. Variability in anatomical structure and chemical composition of cell walls within a stem includes within-ring differences, known as earlywood and latewood (Fig. 2, 3b & 3c), radial variations resulting a

b

c

Figure 1. Macerated Norway spruce tracheids of the last latewood cell row and the adjacent first earlywood cell rows formed in the following growing season (a & b) and tracheids of the earlywood formed later in the growing season (c). Macerated tracheid samples came from young Norway spruce trees (field age 5 years) with different growth characteristics; this explains their differences in size. Hatched bars indicate regions of cross-field pitting. The reference bar represents 200 µm. The figure is modified after Rosner et al. (2007).

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Figure 2. Transverse semi-thin sections (2 µm) of mature Norway spruce earlywood with open (a) and closed (aspirated) bordered pits (b) and of Norway spruce latewood (c) embedded in Technovit 7100 (Heraeus Kulzer GmbH, Wehrheim, Germany) and stained with toluidine blue. Arrows point at the darker stained tori of the pit membranes. Toluidine blue stains the torus dark blue /purple, which gives contrast to the light blue stained pit chamber walls and secondary cell walls of the tracheids (a, b). Bordered pits are merely present at radial cell walls (a, b) but can be as well found in tangential cell walls (c). Reference bars represent 50 µm. Technical details can be found in Rosner et al. (2010).

from cambial maturation and sapwood aging, and differences associated with height position within the trunk (Zobel & Van Buijtenen 1989; Gartner 1995; Lundgren 2004; Havimo et al. 2008; Lachenbruch et al. 2011). A general concept in conifer species such as Norway spruce is that the radial cell wall of a tracheid is thicker than the tangential cell wall (Brändström 2001). Radial width, tangential width and cell-wall thickness vary even along the tracheid length and, moreover, at each contact with rays a sudden change in tangential width and cell wall thickness is observed (Sarén et al. 2001; Neagu et al. 2006). Bordered pits, which are found more frequently in the radial cell walls, can thus be seen as natural irregularities influencing the biomechanical properties of the tracheids (Sirviö & Kärenlampi 1998). Water transport in xylem is achieved under negative pressure: general structural concepts Water is transported from soil to the transpiring leaves under negative hydrostatic pressure (Zimmermann 1983; Richter 2001; Tyree 2003; Domec 2011), requiring high mechanical strength of the tracheid cell walls in order to avoid implosion, and sufficient

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a

b c d

Figure 3. Transverse sections (20 µm) of juvenile wood (1st –2nd annual ring) from the tree top (a, b) and mature wood (17 th –19 th annual ring) from the lower tree trunk (c, d) of Norway spruce. Reference bars represent 50 µm (a, d) and 200 µm (b, c).

safety factors against the breakage of the water column (cavitation). According to the air-seeding hypothesis (Tyree & Zimmermann 2002), cavitation due to drought stress will occur when the pressure difference between water in a conduit and surrounding air exceeds the capillary forces at the air-water interface in the conduit wall. Under these conditions, air will be pulled into the conduit and the air bubble will cause a phase change to vapor. High wood density is supposed to be a common strategy to guarantee low vulnerability to cavitation. In theory, greater resistance to cavitation requires a safer design for resisting implosion, because the cell walls have to withstand higher tensile strain before cavitation occurs (Hacke et al. 2001; Hacke & Sperry 2001). Tensile stresses in a water-filled conduit are supposed to increase with decreasing squared double cell wall (t) to span ratio (b), termed conduit wall reinforcement [(t/b)2], based on the fact that both mechanical strength and stiffness increase with increasing wood density (Hacke

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et al. 2001; Hacke & Sperry 2001; Pitterman et al. 2006; Sperry et al. 2006; Domec et al. 2009). An increase in density, however, goes along with a decrease in hydraulic efficiency, because it is achieved mainly by narrowing lumen diameter rather than increasing cell wall thickness (Hannrup et al. 2004; Pitterman et al. 2006; Sperry et al. 2006). Resistance to drought-induced cavitation is however not directly related to wood density (Hacke et al. 2001; Hacke & Sperry 2001; Domec & Gartner 2002a), but is more closely related to the function of the pit membrane properties (Tyree & Sperry 1989; Sperry & Tyree 1990; Tyree & Ewers 1991; Sperry 1995; Sperry & Ikeda 1997; Hacke & Sperry 2001; Jansen et al. 2012). When sapwood from the lower trunk of older trees is compared to sapwood of very young trees (Rosner et al. 2007, 2008), the relationship between wood density and vulnerability to cavitation actually exists in Norway spruce: P50 (the water potential necessary to cause 50% loss of hydraulic conductivity) increases with decreasing wood density (Fig. 4). However, does the construction of such a relationship help to select individuals with high drought susceptibility or is it helpful to understand strategies or mechanisms behind hydraulic vulnerability? A closer view is necessary to learn e.g. which tracheids are the first to cavitate within an annual ring, if the relationship between P50 and density is valid for wood at a given cambial age and if a similar relationship exists within a tree trunk. -2

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Figure 4. Clonal means of the negative of the air pressure applied by a means of a pressure collar causing 50 % loss of hydraulic conductivity (P50 ) plotted against wood density of juvenile sapwood specimen (filled symbols, annual rings 2–3) of the trunk from 5-year-old trees and mature sapwood specimen (open symbols, annual rings 17–19) from the lower tree trunk of 24-year-old trees. Error bars show one standard error. Description of the study sites and methodological approaches for assessment of P50 can be found in Rosner et al. (2007) for juvenile wood and in Rosner et al. (2008) for mature wood. Wood density was calculated from the volume and the weight of the specimen in the oven-dried state.

Which are the hydraulically most vulnerable tracheids within an annual ring? The efficiency of water transport increases with increasing conduit diameter (Pothier et al. 1989; Lo Gullo & Salleo 1991; Tyree et al. 1994) and as a direct function of variability in the resistance at the pit membrane (Pothier et al. 1989; Hacke et al. 2004, 2006). Within a conifer tree, hydraulic effectiveness and safety should be conflicting xylem functions: the most conductive conduits are supposed to be the most vulnerable ones (Sellin 1991; Cochard 1992; Hacke & Sperry 2001; Domec & Gartner 2001; Tyree & Zimmermann 2002). Norway spruce was the first species where two up-to-date non-destructive testing and analysis methods were applied from technical engineering

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in order to “look inside” dehydrating sapwood in order to test this hypothesis on the within-ring level. Acoustic emission (AE) feature analysis (Rosner et al. 2006) and neutron radiography (Rosner et al. 2012) gave quite new insights into processes associated with movement of free water inside Norway spruce sapwood. The bulk of AE with the strongest frequencies in the range of 100–300 kHz are supposed to be induced by cavitation, i.e. the rapid tension release in the tracheid lumen as liquid water at negative pressure is suddenly replaced by water vapor (Milburn & Johnson 1966; Tyree et al. 1984; Kawamoto & Williams 2002). The plant physiological approach of AE testing has been focused on counting ultrasonic signals surpassing a defined detection threshold, on the assumption that the cumulated number of AE corresponds to a loss in hydraulic conductivity (e.g. Lo Gullo & Salleo 1991; Cochard 1992; Kikuta et al. 2003; Hölttä et al. 2005). The energy of an acoustic signal, a waveform feature that depends on amplitude and signal duration, can give additional information on its source. Rosner et al. (2006) monitored radial dimensional changes together with AE of small dehydrating Norway spruce sapwood beams and performed post-hoc waveform analyses. The mean energies of acoustic signals plotted against time showed a typical pattern (Fig. 5a, d), where high energy AE is produced not right at the beginning but quite early during dehydration. According to the Hagen-Poiseuille equation, cavitation of big diameter tracheids causes much higher hydraulic losses than cavitation of small diameter tracheids. AE feature analysis (Rosner et al. 2006) allows quantifying cavitation events relative to their consequence upon conductivity loss, because AE energy or amplitude increases with increasing tracheid lumen area (Rosner et al. 2009; Mayr & Rosner 2011; Wolkerstorfer et al. 2012). It is hypothesized that bigger tracheids emit stronger AE signals due to cavitation because they are more prone to deformation upon negative pressure than smaller tracheids (Rosner et al. 2009). Dimensional changes in association with negative pressure in cell lumen or capillaries can be observed in sapwood of living stems (Neher 1993; Herzog et al. 1996; Zweifel et al. 2001; Cochard 2001; Offenthaler et al. 2001; Hölttä et al. 2005; Conejero et al. 2007) and also during drying of isolated sapwood specimens (Irvine & Grace 1997; Rosner et al. 2009; Hansmann et al. 2011). Radial dimensional changes of Norway spruce sapwood occur immediately after the drying process starts (Fig. 5b, e). The partial recovery is due to a stress release when it comes to the breakage of the water columns inside the tracheids. When moisture content approaches towards fibre saturation, i. e. when the cell lumen contains no longer free water, but cell walls are fully saturated with liquid (Skaar et al. 1988), the observed recovery process is however overlaid by cell wall shrinkage (Fig. 5c, f). By means of neutron radiography it was validated that dimensional changes of small sapwood beams at moderate moisture loss are not only caused by drying and rewetting of surface layers (Rosner et al. 2012). Neutrons are more sensitive than X-ray beams to some major elements, such as hydrogen, and they are particularly suitable for investigating both wood structure of dry wood as well as free or bound water in the wood (Lehmann et al. 2001; Mannes et al. 2009; Sonderegger et al. 2010). An oven-dry wood specimen still contains about 6% hydrogen, which accounts for 90% of the attenuation of the neutrons (Mannes et al. 2009). Neutron radiography is therefore suitable to investigate structure together with moisture distribution within a wood specimen

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AE Energy /10 min ( pVs)

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Figure 5. Courses of the mean acoustic emission energy/10 min (a, d), the rate of radial dimensional changes /10 min (b, e) and the cumulated radial dimensional changes (c, f ) of dehydrating fresh mature Norway spruce sapwood beams (6 × 6 × 100 mm) plotted against time and against 5% relative water loss steps. Dehydration was performed at ambient conditions (25°C, 30% r. h.). Radial dimensional changes and acoustic emission were assessed with a load cell and a resonant 150 kHz acoustic transducer positioned on the tangential wood surface of a fully saturated specimen by means of a self-designed acrylic resin clamp assemblage which is described in detail in Rosner et al. (2009). Error bars (n = 12) show one standard error.

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(Sonderegger et al. 2010). By means of this imaging technique, Rosner et al. (2012) could prove that free water in the middle part of a small Norway spruce sapwood beam is not continuously removed from the outer to the inner parts but earlier from low density earlywood than from the main latewood parts. In the transmission profiles (Fig. 6), moisture loss is indicated by an increase in transmission. Quite unexpected was, that Position (mm)

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Figure 6. Transmission profiles of a Norway spruce sapwood beam with wide annual rings (a) and narrow annual rings (b) at different stages of relative moisture loss (RWL). Below each transmission profile the transverse section of the wood beam with dimensions at full saturation is shown. The x-axis represents the positions of the transmission profiles from both sides of the middle part of the beam (= 0 mm), since the specimen shows dimensional changes upon dehydration. The figure is modified after Rosner et al. (2012), where the methodological approach is described in detail.

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not all latewood tracheids were less prone to cavitation, since transmission profiles gave strong evidence that some latewood tracheids lost free moisture earlier than earlywood tracheids. In the middle part of the specimens, where shrinkage is negligible (position around 0 mm), transmission after 20% relative moisture loss was higher in latewood than in earlywood (Fig. 6b). Domec and Gartner (2002b) and Domec et al. (2006) reported similar findings for Douglas-fir sapwood. Latewood-bordered pits of Norway spruce do not aspirate upon dehydration (Liese & Bauch 1967) giving strong evidence that different mechanisms of air seeding exist in earlywood and latewood (Rosner 2012). The rigidity of the pit membrane is supposed to have an impact upon the mechanism how air seeding occurs (Gartner 1995; Domec & Gartner 2002b; Hacke et al. 2004; Domec et al. 2006, 2008). Pits act as valves, preventing the spread of bubbles through the conducting system (Tyree & Zimmermann 2002). Entry of air occurs either when the torus is not tightly sealed against the overarching pit border or when air bubbles are pulled through small pores in the torus or, in non-aspirated pits, through the margo of the pit membrane (Sperry & Tyree 1990; Tyree & Zimmermann 2002; Domec & Gartner 2002b; Choat & Pitterman 2009; Cochard et al. 2009; Delzon et al. 2010; Jansen et al. 2012). The strategy of Norway spruce earlywood to prevent air seeding is thus assumed to be pit aspiration (Fig. 2b), whereas latewood tracheids invest in rigid pit membranes with thicker margo strands and smaller pores. Air seeding in latewood will thus take place directly through the margo as hypothesized for Douglas fir (Domec & Gartner 2002b). Some latewood tracheids, probably those with the smallest lumen diameters, should however remain conductive even at low negative pressures, since they bear the smallest margo pores (Domec et al. 2006). To sum up, Norway spruce earlywood is more prone to cavitation than the main latewood parts. Some latewood tracheids are however highly vulnerable to cavitation, probably because their pits do not aspirate and pit membranes are not densely enough structured to avoid air entry. Whether cavitation of such tracheids has a high impact on conductivity loss should be a topic of further research. Hydraulics and mechanics of the mature trunkwood: mean ring density is a predictive trait Rosner et al. (2008) investigated the impact of growth and basic density on hydraulics and mechanical properties of six different Norway spruce clones. For the study, clones with different diameter and height growth were selected. The same clones were grown on two sites with different water availability in southern Sweden. Basic density, hydraulic, and mechanical parameters in mature spruce wood varied considerably between clones (Fig. 7), suggesting high breeding potential for these parameters (Rozenberg & Cahalan 1997; Hannrup et al. 2004). Stem wood of rapidly growing clones had significantly lower basic density, was more vulnerable to cavitation (Fig. 7a) and had higher values of sapwood area specific hydraulic conductivity at full saturation (Fig. 7b). Rapidly growing clones produced however trunkwood with lower bending strength and stiffness (modulus of elasticity, Fig. 7c) and compression strength and stiffness in the axial direction (Young’s modulus). In accordance with Hacke et al. (2001), a clear tradeoff existed between hydraulic conductivity and vulnerability to cavitation, where

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spruce clones with high basic density had significantly lower hydraulic vulnerability but also lower hydraulic conductivity at full saturation and thus less rapid growth. As mentioned above, the relationship between hydraulic performance and density is not a direct one, but depends on the characteristics of the bordered pits. Density is a quite good indirect predictive trait for hydraulic vulnerability (Fig. 7d), hydraulic conductivity at full saturation (Fig. 7e) and bending stiffness (Fig. 7f) in mature spruce wood. However, empirical models for predicting hydraulics or mechanics from basic density have to take account for site effects or forestry practices influencing

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9 8 7

r2 wet = 0.96 r 2 dry = 0.71

6 0



0

8

10

12

DBH (cm)

14

r2 wet = 0.94 r 2 dry = 0.76 r 2 = 0.72

16 0.00 0.34 0.36

0.38 0.40

0.42 0.44

BD (g cm -3 )

Figure 7. Clonal means of the diameter at breast height (DBH) and basic density (BD) as functions of clonal means of the negative of the air pressure applied by a means of a pressure collar causing 50% loss of hydraulic conductivity (P50), the specific hydraulic conductivity at full saturation (ks100), and the bending stiffness (MOE). Filled symbols denote trees from the wetter site (Tönnersjöheden); open symbols denote trees from the drier site (Vissefjärda); each symbol denotes a different clone. Error bars represent one standard error. Significant linear (a, b, c, d, f) and quadratic (e) relationships (P < 0.05) are indicated by solid regression lines for the wet site (n = 6) and by dashed regression lines for the dry site (n = 6). Dotted lines indicate significant linear (a, b, c, d, f) and quadratic (e) relationships across sites (n = 12). The figure is modified after Rosner et al. (2008), where detailed descriptions of clones, sites and methods can be found.

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the physiological stage of the individual tree, e.g. of different crown lengths or stem taper (Deleuze et al. 1996; Kantola & Mäkelä 2004; Jaakkola et al. 2005). Although maximum hydraulic conductivity was negatively related to mechanical strength and stiffness on both sites and across sites, Rosner et al. (2008) found hints for smart structural solutions for achievement of both high hydraulic efficiency (the sapwood area-specific conductivity) and mechanical strength: clones growing on the wetter site showed significantly higher hydraulic conductivities and thus higher growth (Fig. 7e), but also higher mechanical strength and stiffness (Fig. 7f) than those on the drier site. Higher mechanical stiffness was not achieved by increasing mean ring density. Hydraulic efficiency and mechanical stability in the axial direction can be obviously obtained –to some extent– simultaneously. Such strategies are described in the chapter “Structural tradeoffs due to different hydraulic and mechanical demands within the trunk”. The genetic determination of wood density in Norway spruce is however generally high (Rozenberg & Cahalan 1997; Hannrup et al. 2004; Jaakkola et al. 2005). It is therefore suggested that also mechanical properties and vulnerability to cavitation might be under strong genetic control (Rosner et al. 2008). In species such as Norway spruce with growth rates inversely related to specific gravity (Herman et al. 1998; Hannrup et al. 2004; Steffenrem et al. 2009), rapid growth rate is the principal criterion for tree breeding and high density only second (Zobel & Jett 1995). Selecting for growth may thus lead not only to a reduction in mechanical strength and stiffness but also to a reduction in hydraulic safety if adequate precautions are not taken (Booker & Sell 1998; Domec & Gartner 2002a; Rozenberg et al. 2002; Cochard et al. 2007). Hydraulic and mechanical demands along a tree trunk: Compromises are necessary Natural selection has optimized wood structure within tree trunks to maximize survival of the species. The optimum structures for each biological wood function will most likely differ, leading to conflicting demands on wood structure for physiological fitness (Schniewind 1962; Baas 1983; Gartner 2001). Within a conifer tree trunk, structural, hydraulic and biomechanical characteristics show therefore a higher variability than can be found in mature trunkwood of different individuals from the same species summarized as the concept of juvenile wood, or more correct “core wood” (Gartner 1995; Lachenbruch et al. 2011). Juvenile Norway spruce wood is produced from cambial zones younger than 15–20 years (Kučera 1994; Saranpää 1994; Lindström et al. 1998) and is characterized by shorter cells with thinner cell walls and smaller lumen diameters (Fig. 3a, d), larger microfibril angles (the deviation of the microfibrils in the S2 layer from the tracheid axis), different specific gravity and by within-ring density variations (Fig. 3b, c) compared to mature wood (Saranpää 1994; Lindström 1997; Saranpää et al. 2000; Sarén et al. 2001; Jungnikl et al. 2008). Kučera (1994) found that the formation of mature Norway spruce wood on the lower trunk commences when the annual height growth has culminated. Mechanical demands within a conifer trunk with special reference to Norway spruce Tree trunks experience short- and long-term mechanical stresses from a variety of causes such as gravity, wind, weight of snow, removal of a branch, partial failure of the anchorage system, or growth and development (Gartner 1995; Lachenbruch et al.

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2011). The weight of a tree will subject the trunk largely to bending, but winds will normally cause twisting as well, because neither has a tree a completely symmetrical shape, nor is wind load uniform (Vogel 1995). Growth stress in the older wood develops along the tree axes in radial, tangential and axial direction due to sequential production of annual rings (Archer 1986; Gartner 1995). Where stems are out of their equilibrium positions due to static and dynamic forces of gravity and wind loads, compression wood is formed on the lower side of leaning stems or opposite to the windward side (Archer 1986; Timell 1986; Telewski 1995; Mattheck 1998; Spatz & Bruechert 2000). Spiral grain, i.e. the angle between stem axis and the inclination of the longitudinal tracheids in the direction of the wind-induced torque offers an additive advantage of the tree to cope with heavy wind loads (Skatter & Kučera 1997). Spiral-grained Norway spruce stems bend and twist more when exposed to strong winds, offering less wind resistance and being less likely to break. Moreover, through the bending and twisting, snow can slide down from branches rather than breaking them (Kubler 1991). Structure-function relationships within the main trunk presented hereafter are dealing with normal or opposite knot-free Norway spruce wood and mechanical testing was performed along or normal to the grain. Rosner and Karlsson (2011) investigated bending stiffness of the outer sapwood within the trunk of 24-year-old Norway spruce trees. Bending stiffness was found to be lower at the tree top than at the lower tree trunk (Fig. 8e). Higher bending stiffness at the base than towards the top should provide several advantages for the stability of a Norway spruce tree, e.g. when it interacts with strong winds (Kubler 1991; Lundström et al. 2007). Bending resistance is highest where it is most needed (Mattheck 1998), thus higher up in the more tapered stems (Milne & Blackburn 1989). High flexibility of the upper part prevents the crown from catching wind flow and heavy snow loads are allowed to slip down from the crown rather than breaking it (Kubler 1991). Hydraulic demands within Norway spruce trunks Within a conifer trunk, hydraulic efficiency has been reported to increase from pith to bark, i.e. from juvenile to mature wood (Domec & Gartner 2001; Spicer & Gartner 2001; Domec & Gartner 2002a; Domec et al. 2009). Generally, wood higher up in the trunk (higher amount of juvenile wood) is modified to be more resistant to cavitation than the wood at the base because more negative pressures develop higher up in the crown (Cochard 1992; Domec & Gartner 2001; Domec & Gartner 2002a; Domec et al. 2009). These relationships proved valid as well for Norway spruce trunks; young trunkwood from the tree top had lower hydraulic vulnerability (Fig. 8a) as well as lower specific hydraulic conductivity than mature trunkwood of the lower trunk (Rosner et al. 2006; Rosner & Karlsson 2011; Wolkerstorfer et al. 2012). Higher hydraulic efficiency was achieved by higher hydraulic lumen diameters (Anfodillo et al. 2005, 2013; Domec et al. 2009) and most likely by modifications of the characteristics of bordered pits (Domec et al. 2006, 2008). Higher hydraulic vulnerability is aligned with lower wall/lumen rations in earlywood tracheids (Fig. 8a) (Domec et al. 2009), thus with a higher susceptibility to radial deformation (Fig. 8c) under a given (negative) pressure (Hacke et al. 2001; Hacke & Sperry 2001; Rosner & Karlsson 2011).

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In accordance with Domec et al. (2009), who performed investigations on Douglas fir and Ponderosa pine, hydraulic vulnerability was not related to mean ring density within the main Norway spruce trunk: juvenile wood from the tree top showed similar density values as mature wood at breast height (Rosner & Karlsson 2011). Moreover, in pine species mean ring density was found to be even lower in juvenile than in mature trunkwood (Domec et al. 2009). Acoustic energy 50% (MPa)

5

d

a

4 r 2 = 0.44 3

2 0 16

r 2 = 0.72

b

MOE (GPa)

e

r 2 = 0.70

14 12

r 2 = 0.80

10 8 6 4

Peak force σr (MPa)

0

6

4

2 0 0.00



f

c

8

r 2 = 0.73 0.03 0.04 0.05

r 2 = 0.46 0.06

Wall reinforcement (t/b) 2

5

10

15

20

25

Latewood (%)

Figure 8. Hydraulic and mechanical traits related to anatomical properties. Vulnerability to cavitation assessed by the acoustic method (a, d), modulus of elasticity in bending (b, e) and compression strength (c, f ) plotted against the conduit wall reinforcement and latewood percentage of juvenile (closed circles) and mature (open circles) Norway spruce sapwood specimens. Significant linear relationships across cambial age are indicated by solid regression lines, the significant relationship in mature wood by a broken regression line. Material and methods are described in detail in Rosner & Karlsson (2011).

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Structural tradeoffs due to different hydraulic and mechanical demands within the trunk An increase in density goes along with an increase in mechanical strength but also with a decrease in hydraulic efficiency, because it is achieved mainly by narrowing lumen diameter rather than increasing cell wall thickness (Hannrup et al. 2004; Pitterman et al. 2006; Sperry et al. 2006). Bending strength and axial compression strength, however, can be modified by varying the percentage and density of latewood (Hacke & Sperry 2001; Pitterman et al. 2006; Sperry et al. 2006; Jyske et al. 2008; Domec et al. 2009), fibre length (Mencuccini et al. 1997; Ezquerra & Gil 2001), arrangement of the cell wall layers (Jagels et al. 2003; Jagels & Visscher 2006), microfibril angle (Meylan & Probine 1969; Booker & Sell 1998; Lichtenegger et al. 1999; Evans & Ilic 2001; Jungnikl et al. 2008; Salmén & Burgert 2009) or cell wall chemistry (Gindl 2001, Gindl 2002; Kukkola et al. 2008). Moreover, hydraulic efficiency can be increased by increasing the pit pore size in earlywood (Mayr et al. 2002; Rosner et al. 2007). Within a tree trunk, hydraulic efficiency and bending strength as well as axial compression strength are therefore not necessarily conflicting wood functions (Mencuccini et al. 1997; Jagels & Visscher 2006). Accordingly, mature Norway spruce trunkwood showed higher hydraulic efficiency (hydraulic conductivity at full saturation) and bending stiffness than juvenile wood from the tree top, although both wood types had similar mean ring densities (Rosner & Karlsson 2011). Mean ring density in Norway spruce decreases till annual ring eight and increases again thereafter (Kučera 1994), which is most likely achieved by variations in latewood percentage or latewood density (Mäkinen et al. 2002; Jyske et al. 2008). Accordingly, high bending stiffness across cambial age within Norway spruce trunks was strongly related to higher latewood percentage (Fig. 8e). An increase with tracheid length goes along with an increase in bending stiffness as well as in hydraulic efficiency (Mencuccini et al. 1997; Ezquerra & Gil 2001) and is known to increase with cambial age in Norway spruce (Schultze-Dewitz 1959; Saranpää 1994; Lindström 1997; Sirviö & Kärenlampi 2001). High microfibril angles in the S2 layer of the cell wall of juvenile Norway spruce wood (Lindström et al. 1998; Brändström 2001; Hannrup et al. 2004) guarantee higher flexibility of the upper crown (Neagu et al. 2006) but should have no negative implication upon vulnerability to cavitation. However, does Norway spruce trunkwood also manage to guarantee high mechanical strength perpendicular to the grain and high hydraulic efficiency? Rosner and Karlsson (2011) were the first to relate compression strength perpendicular to the grain to anatomical and hydraulic parameters within a conifer trunk. Hydraulically less safe mature Norway spruce sapwood from the older trunk had higher specific hydraulic conductivity and bending stiffness (Fig. 8e), but lower radial compression strength (Fig. 8c) and conduit wall reinforcement (Fig. 8b) than hydraulically safer juvenile wood from the young crown. A clear tradeoff existed between hydraulic efficiency and compression strength perpendicular to the grain, since both traits are strongly related to the characteristics of the “weakest” wood parts, the low density earlywood (Müller et al. 2003). Structural compromises such as increasing conduit wall reinforcement in mature earlywood would probably be too costly for the tree. Radial compression strength (defined as the first peak in the stress/strength curve)

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is therefore negatively correlated to bending stiffness within a Norway spruce trunk (Rosner & Karlsson 2011). It is proposed that radial compression strength could be a highly predictive parameter for the resistance against vulnerability to cavitation, based on the close relationship to conduit wall reinforcement (Fig. 8c). These relationships should be tested in further studies either within one species or across species. Hydraulic and mechanical demands of young Norway spruce stems A young/small tree is in competition with others for light, water and nutrient supply but has a shallow root system and little water storage capacity (Lachenbruch et al. 2011; Scholz et al. 2011) which implies the need for higher hydraulic safety factors than are necessary in mature trunkwood of old-growth trees (Domec et al.2009). In species such as Norway spruce that manage to survive at the alpine timberline (Mayr et al. 2002, 2003) or in northern regions (Tollefsrud et al. 2008), the young trunks must as well be highly flexible in order to carry heavy wind and snow loads. Slight changes in stem orientation due to competing for light together with the important biomechanical task of keeping the stem in the upright position result in variable amounts of compression wood (Lindström et al. 1998; Zobel & Sprague 1998; Lachenbruch et al. 2011). Compression wood, usually present on the lower side of leaning stems or opposite to the windward side, has thicker tracheid cell walls and smaller tracheid lumen than opposite or “normal ”wood. Tracheids of compression wood have a more rounded shape in the transverse plane and their cell walls contain more lignin and have higher S2 microfibril angles than those of “normal” wood (Timell 1986; Gorisek & Torelli 1999; Bergander et al. 2002; Burgert et al. 2002; Gindl 2002). Norway spruce compression wood has a much higher density than normal wood but has also a higher vulnerability to cavitation (Mayr & Cochard 2003). Structure-function relationships are thus much more complex in the trunk of young/small spruce trees than in (normal) mature trunkwood because a density based tradeoff in hydraulic functions can be masked by the individual mechanical demands (Mayr et al. 2003). Rosner et al. (2007) investigated structure-function relationships in 2–3-year-old stem segments from eight different Norway spruce clones (field age five years) differing in growth characteristics. Ring width, wood density, latewood percentage, lumen diameter, wall thickness, tracheid length and pit dimensions of earlywood cells, spiral grain and microfibril angles were tested together with hydraulic and mechanical traits. Vulnerability to cavitation (P50, Fig. 4) and specific hydraulic conductivity of trunkwood from trees younger than five years were found to be much lower compared to mature trunkwood or wood from the tree top of older spruce trees (Rosner et al. 2006), which might be explained by limited access to ground water due to a less efficient root system. Other than in mature trunkwood, wood density was not related to the hydraulic vulnerability parameters assessed (Mayr et al. 2003), which comprised P12 and P50, the pressure that is necessary to result in 12% and 50% loss of hydraulic conductivity, respectively, and PLC4MPa, the percent loss of conductivity induced by 4 MPa pressure. Traits associated with higher hydraulic vulnerability in the young tree trunk were long tracheids (P12) and thick earlywood cell walls (PLC4MPa). The positive relationship between earlywood wall thickness and vulnerability to cavitation suggests that air

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seeding through the margo of the bordered pits may also occur in juvenile Norway spruce earlywood (Domec & Gartner 2002b; Jansen et al. 2012). Pit membranes of earlywood cells with thicker walls may be less flexible, so they cannot be that easily deflected to seal off the pit aperture completely (Sperry & Tyree 1990; Domec et al. 2006, 2008) as it is characteristic for compression wood (Mayr & Cochard 2003) and conifer latewood (Domec & Gartner 2002b). One of the most interesting findings of Rosner et al. (2007) was that maximum hydraulic conductivity in young Norway spruce trunks is not only positively related to tracheid length and pit dimensions (Mayr et al. 2002) but also strongly to spiral grain. Spiral grain might offer an additional advantage for reducing flow resistance of the bordered pits of the first formed earlywood tracheids, which are characterized by rounded tips and a quite uniform distribution of pits along the entire length (Fig. 1a, b), as found in light bands of branch compression wood (Mayr et al. 2005). In earlywood formed later in the growing season, bordered pits achieving axial water flow can be found exclusively near the tapered tracheid ends (Fig. 1c). Hydraulic conductivity and vulnerability to cavitation, estimated as PLC4MPa, showed only a weak tradeoff; both traits reached however higher values in trees with fast growth. Variability in mechanical properties (bending strength and stiffness, axial compression strength and stiffness) of the young tree trunk depended mostly on wood density, but also on the amount of compression wood (Rosner et al. 2007). A density-based tradeoff between hydraulic characteristics and mechanical strength or stiffness (Hacke et al. 2001; Hacke & Sperry 2001; Domec & Gartner 2002a; Bouffier et al. 2003) is probably masked by structural compromises associated with mechanical demands of the young trunk (Mayr et al. 2002). High wood density in normal wood or compression wood can be compensated for flow reduction by a higher pit frequency in the first formed earlywood tracheids (Mayr et al. 2005). As mentioned above, mechanical support can be achieved not only by increasing mean ring wood density. Moreover, a proposed density-based tradeoff in hydraulic parameters is additionally masked by the fact that compression wood has a higher density than normal wood but is more vulnerable to cavitation (Mayr & Cochard 2003; Mayr et al. 2005). Mayr et al. (2003) measured in leader shoots of small Norway spruce trees from the alpine timber line a 1.4 times higher specific hydraulic conductivity as well as a 4.9 fold higher leaf specific conductivity than in side twigs, although vulnerability to cavitation was much lower in the former. They also found that lower vulnerability to cavitation is not related to wood density, expressed as the wall/lumen ratio (Hacke et al. 2001; Hacke & Sperry 2001), but correspond to smaller pit dimensions. Genetic determination of hydraulic vulnerability was found to be quite weak in young stem segments, but its predictive structural traits were under strong genetic control (Rosner et al. 2007). Most of these traits, such as tracheid length and pit aperture percentage, were positively related to growth. The positive relationship between density and hydraulic safety (Hacke & Sperry 2001) will become more apparent in mature Norway spruce wood (Rosner et al. 2008); early selection for high growth (Zobel & Jett 1995) could thus result in individuals with increased sensitivity to cavitation in the mature trunkwood (Domec & Gartner 2002a).

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CONCLUSIONS AND OUTLOOK

New applications in tree physiological research such as neutron radiography and acoustic emission feature analysis together with assessment of dimensional changes during dehydration helped to fill some gaps in our knowledge on hydraulic vulnerability within an annual ring of Norway spruce trunkwood. A future task in this regard is to perform anatomical and chemical analyses of cell walls and pit membranes in order to understand why some latewood compartments are more vulnerable to cavitation than low density earlywood. It was also necessary to investigate to what extent cavitation of highly vulnerable latewood tracheids contributes to conductivity loss since in mature Norway spruce wood, mean ring density is linked to hydraulic performance. Relating selected parameters from within-ring X-ray density profiles to vulnerability traits was the next step in our search for easily assessable parameters for estimating hydraulic performance under drought stress. Up till now we know that sapwood of faster growing Norway spruce clones is more sensitive to cavitation and to mechanical stresses along the grain due to lower mean ring density; however, refinement of empirical models is still necessary. A precondition for using wood density parameters to screen for high hydraulic safety is taking account for structural compromises associated with mechanical demands within a tree trunk, e.g. guaranteeing high flexibility of the tree top. Thus, only anatomical data assessed in tree rings of similar cambial age sampled at defined (relative) distance either from the tree top or from the ground will give reliable results on hydraulic performance. Structural compromises and complex wood structure, where a density-based tradeoff between hydraulic characteristics and bending stiffness is masked by the presence of reaction (compression) wood is probably the main reason why density is no useful trait for predicting hydraulic vulnerability in small Norway spruce trees. Thus, more research on reliable predictive traits for hydraulic safety of young juvenile wood is needed. We should be aware that Norway spruce faces an uncertain future under the influence of continued global warming. Gaining more knowledge on structure-function relationships within tree trunks will enable the development of easily assessable and fast methods to estimate a tree’s susceptibility to drought stress and will thus help selecting more suitable provenances or clones. An important task should be as well the investigation of refilling processes in Norway spruce sapwood, since a recent study points out that embolism repair is a key trait for the characterization of the strategy of a species to cope with drought stress (Johnson et al. 2012). ACKNOWLEDGEMENTS This review was financed by the Austrian Science Fund (FWF): V146-B16.

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Prislan IAWA Journal et al. – Changes 34 (4), 2013: in the391– cambium 407

Review of cellular and subcellular changes in the cambium Peter Prislan1,2,*, Katarina Čufar 2, Gerald Koch3, Uwe Schmitt 3 and Jožica Gričar1 1Slovenian

Forestry Institute, Večna pot 2, SI-1000 Ljubljana, Slovenia Faculty, Department of Wood Science and Technology, University of Ljubljana, Rožna dolina, Cesta VIII/34, SI-1000 Ljubljana, Slovenia 3 Thünen Institute of Wood Research, Leuschnerstraße 91, D-21031 Hamburg, Germany *Corresponding author; e-mail: [email protected]

2 Biotechnical

ABSTRACT

The commonest approach to studying cambial productivity is conventional light microscopy, which is widely used in wood formation studies. The number of such studies has increased rapidly in the past decade, usually in order to elucidate the relationship between growth and environmental factors. However, some aspects of cambial seasonality are often overlooked or neglected. Observations with transmission electron microscopy provide a more detailed insight into changes occurring on the ultra-structural level in cambial cells. Criteria for defining cambial activity are not yet fully clarified, especially when observing it at different resolutions, i. e., on cellular, subcellular and ultrastructural levels. The goal of this review is to contribute to clarification of the terms mainly used, such as cambial dormancy, reactivation, activity, productivity and transition between different states, resting period and quiescence, which describe structural modifications of cambial cells during the various phases of their seasonal cycle. Based on our own cambium observations on adult beech trees growing at two different elevations, which were made with light and transmission electron microscopy, we discuss the influence of weather conditions on cambial activity and the advantage of the complementary use of different techniques and resolutions. Keywords: Cambial activity, cambial cells, Fagus sylvatica, light microscopy, transmission electron microscopy, ultrastructural changes. INTRODUCTION

Growth in plants takes place in specialised tissues, so-called meristems, which act as central control points for growth and development, receiving, integrating, responding to and broadcasting growth-regulating signals (Risopatron et al. 2010). Two meristems are responsible for the growth of trees: apical (growth in length) and lateral (growth in girth) (Lachaud et al. 1999). Apical meristems produce primary tissues, whereas lateral meristems (i.e., vascular and cork cambium) contribute to the production of secondary tissues (Mauseth 2009). The vascular cambium develops from the procambium, which in turn is derived from parenchyma cells that have regained the capability to divide © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000032

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(Evert 2006). The cambium is a bifacial meristem because it produces xylem cells in the centripetal and phloem cells in the centrifugal direction (Larson 1994). Cambial activity ensures the perennial life of trees through regular renewal of functional xylem and phloem (Plomion et al. 2001). Moreover, cambial growth might be considered to be the tree’s way of ensuring that its stem and branches have sufficient structural support and hydraulic conductivity to grow against gravity, while also providing for the needs of the root system (Savidge 2000b). The annual course of cambial activity is generally related to the alternation of cold and warm or dry and rainy seasons (Lachaud et al. 1999). In some climatic regions, i.e. tropics, cambial activity may continue throughout the year, whereas in temperate regions the activity of the cambium is usually periodical, subjected to the tree’s internal regulation (genetic and hormonal) (Ursache et al. 2013) and environmental factors, such as temperature, precipitation, photoperiod and other biotic and abiotic influences (e.g. Wodzicki 2001; Evert 2006; Begum et al. 2013). Commonly, xylem production represents the major proportion of the tree’s radial growth. In addition to the economic importance of wood, and partly also bark, wood increments provide an integral archive of factors affecting its formation before and/or during the time of cambial growth. Dendroclimatological and dendroecological studies, in combination with intra-annual observations of radial growth of trees, are thus useful in order better to understand climate-growth relationships (Čufar et al. 2008; Callado et al. 2013; Costa et al. 2013). Although numerous investigations have been dedicated to the annual rhythm of cambial activity in various tree species, criteria for determining its activity are still not satisfactorily defined, especially in the case of observations (and their comparisons) at different levels, e.g., cellular, sub-cellular and ultra-structural levels (Frankenstein et al. 2005; Prislan et al. 2011; Rathgeber et al. 2011). When observing ultrastructure of cambial cells, for example, the onset of cambial activity can be easily defined when first mitotic figures and phragmoplasts are observed (Larson 1994; Farrar & Evert 1997). However, changes in the ultrastructure of cells, which could also be considered as activity, occur before formation of phragmoplast, as a transition from the dormant to the active state (Farrar & Evert 1997). In wood formation studies, phragmoplasts or mitotic figures cannot be observed due to different methodologies of sample preparation and observation. Therefore reactivation of the cambium is defined by an increase in number of cambial cells and the occurrence of newly formed xylem and phloem cells in early developmental stages (e.g., Gričar et al. 2006; Deslauriers et al. 2008). Rathgeber et al. (2011), who studied cambial phenology and xylem formation in Abies alba, pointed out that the number of cells in the cambium cannot be a precise indicator for its activity, because the increase in cell number may be insignificant. Consequently, the appearance of first enlarging xylem cells was considered as a more appropriate indicator for cambial reactivation (Rathgeber et al. 2011). In contrast, Frankenstein et al. (2005) observed that initial earlywood vessels in ring porous Fraxinus excelsior were formed in the previous growing season and overwintered, and then started to differentiate prior to the onset of cambial cell divisions in spring. Aims of this review are to: (i) highlight the latest findings on structural modifications in cambium that are related to its seasonal activity and cell production; (ii) exemplify

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them by our most recent observations on seasonal cellular and ultrastructural changes in cambium (Prislan et al. 2011) and phenological variation in cambial productivity (Prislan et al. 2013) carried out in Fagus sylvatica growing under different weather conditions and (iii) emphasise the differences in observations at the cellular and ultrastructural level in order to suggest a suitable terminology for determining the onset of cambial activity at different observational levels. Structure and function of cambium

The cambium is composed of highly vacuolated meristematic cells organised in radial files, which give rise to the secondary xylem and phloem. Theoretically, each radial file contains one initial cell, which remains in the meristem, as well as phloem and xylem mother cells, which are produced by the division of cambial initials (Larson 1994). Consequently, the term “cambium” is used to refer to cambial initials and “cambial zone” to the region of cambial initials and mother cells (Lachaud et al. 1999). Since the initials and mother cells of cambium are distinguished cytologically only by a small difference in length, most published data do not distinguish between the two cell types (e.g., Larson 1994; Savidge 2000b). The terms “cambial cells” and “cambium” will be used hereafter to denote all undifferentiated cells capable of division. Savidge (2000a) argued that every cambial cell could be equally competent and that cambium is maintained in response to basipetally transported auxin in conjunction with physical forces. The differential behaviour of cambial cells could be explained by changes in the micro-environment experienced by the genome within each cell (Savidge 1996). While the main function of the cambium is cell division and setting out patterns for differentiation, similar to other meristems, several aspects are unique to vascular cambium. Unlike apical meristems, cambium is a complex tissue containing two morphologically distinct cell types: axially elongated fusiform cambial cells and somewhat isodiametrical ray cambial cells. These cells give rise to the axial and radial cells of the secondary xylem and phloem. The identity of cambial cells is determined by positional cues rather than by cell lineage, because inter-conversion between fusiform and ray cambial cells is a common phenomenon (e.g. Larson 1994; Mellerowicz et al. 2001). The balance in the number and distribution between fusiform and ray cambial cells is maintained by anticlinal divisions and by conversion of one kind of cell into the other. Fusiform cambial cells thus give rise to new cambial ray cells through transverse or oblique divisions, while ray cells elongate into fusiform ones through intrusive growth (Lachaud et al. 1999). Anticlinal (= radial and pseudotransverse), often also referred to as multiplicative, cell divisions ensure the increase in girth of the cambium (Fig. 1). Transverse and anticlinal divisions ensure the maintenance of cambial integrity, while periclinal (= tangential) divisions (also referred as additive divisions) give rise to new cells of xylem and phloem tissue (Larson 1994; Lachaud et al. 1999). Most divisions of fusiform cambial cells are periclinal, in which new cells are added within a radial file towards either the secondary xylem or the secondary phloem (Catesson 1994). In relation to the functioning of the cambium, two aspects appear to be important for the cambial cell production: 1) the number of dividing cambial cells

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Figure 1. a: Transverse and b: radial sections of cambium and parts of the youngest phloem and xylem growth rings in beech (Fagus sylvatica). c: Periclinal or additive (P) and anticlinal or multiplicative (A) division in cambium. — Scale bars = 100 μm.

and how fast the newly formed derivatives are released from the meristematic regio and 2) the duration of the cell cycle. Both aspects may be individually targeted to maximise the rate of cell production on the xylem and phloem sides (Uggla et al. 1998; Mellerowicz et al. 2001). Wood and phloem formation are not predetermined processes but are very plastic expressions of interactions between genotype and the environment (Savidge 2000b) and require positional information that coordinates the radial pattern of the developmental zones (Uggla et al. 1998). Both phloem and xylem are complex tissues, each containing more than one cell type, so cambial derivatives pass through successive stages of differentiation during the development of phloem or xylem (Savidge 2000b). Cell

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differentiation thus involves four major steps: cell expansion, followed by the ordered deposition of a thick multi-layered secondary cell wall and, in the case of sclerenchyma and tracheary elements, also cell wall lignification and cell death. Cambial cell production is normally more active on the xylem side, explaining the considerable disproportion existing between phloem and xylem tissue (Plomion et al. 2001). Fromm (2013) reported that most tree species have xylem to phloem ratios of between 4:1 and 10:1. SEASONAL CHANGES IN CAMBIAL CELLS

In trees of temperate and cold climatic regions, cambial activity is seasonal and depends on a complex of interactions among intrinsic and extrinsic factors (Savidge 1996; Evert 2006). The dormant period starts immediately after the cessation of meristematic activity and lasts until the resumption of cell divisions (Lachaud et al. 1999). Winter dormancy is usually divided into two periods: 1) the resting period or physiological dormancy, which is driven endogenously and 2) quiescence or environmental dormancy, driven by environmental factors (Riding & Little 1984). During the first 2 to 4 weeks of dormancy, the cambium is unable to produce new cells, even when conditions are favourable (resting period). The resting period terminates when the cambium gradually regains the ability to produce new xylem and phloem cells due to favourable environmental conditions (quiescent stage of dormancy).

Rest or physiological dormancy. First 2 to 4 weeks of dirmancy maintained by conditions within the plant (Riding & Little 1984; Rensing & Samuels 2004; Begum et al. 2013).

Quiescence or environmental dormancy controlled by environmental conditions, involves structural, histochemical and functional changes in cambial cells (Riding & Little 1984; Rensing & Samuels 2004; Begum et al. 2013).

According to Farrar & Evert (1997) as well as Lachaud et al. (1999) transition describes cytoplasmic changes in cambial cells (e.g. fusion of large vacuoles, ER becomes roughly surfaced, etc.). Prislan et al. (2011) concluded that with transmission electron microscopy it is possible to follow ultrastructural changes (activities) in cambial cells during the period before the number of cells in cambium increase.

Reactivation - Onset of cell plate formation in cambial cells (Begum et al. 2013).

Histologically (with LM), reactivation of cambium was defined by an increased number of cambial cells – thus we can observe cambial productivity (Prislan et al. 2011).

Figure 2. Schematic presentation of most commonly used terminology (proposed by various authors) in terms of cambial dormancy and activity, for the example of Fagus sylvatica trees growing at 400 m a. s. l. in Slovenia.

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The transition from resting to quiescence involves structural, histochemical and functional changes in cambial cells (Lachaud et al. 1999; Begum et al. 2013). In addition, this transition between the two phases differs significantly among species (Farrar & Evert 1997b; Lachaud et al. 1999). Figure 2 shows the most commonly used terminology in relation to the dormancy and activity of the cambium on the example of Fagus sylvatica. The ultrastructure of cambium cells differs significantly in its active and dormant state and can be seen in the different organisation, distribution, number and shape of the organelles (Farrar & Evert 1997b; Lachaud et al. 1999; Rensing & Samuels 2004) (Fig. 3). Most obvious are the differences in shape and size of the vacuoles, which are small, round, numerous and within a dense cytoplasm in dormant cells (Farrar & Evert 1997a; Rensing & Samuels 2004; Frankenstein et al. 2005; Prislan et al. 2011). In general, the first divisions in spring occur at the end of a 1 to 4 week period characterised by changes in the vacuolar system following the resumption of cyclosis, i. e., the elongation of small vacuoles and their progressive fusion into one or two large vacuoles. Dividing cambial cells contain, among other elements, large vacuoles, rough ER (endoplasmic reticulum), numerous dictyosomes, which produce vesicles and lack storage products such as lipid droplets. The transition from activity to dormancy involves processes whereby large vacuoles fragment into a number of smaller ones, which intersperse throughout the cytoplasm. Rough ER is replaced by smooth ER and an accumulation of storage products takes place, the nature of which depends on the species (Fig. 3). Dictyosomes become fewer and mainly inactive (Farrar & Evert 1997a,b; Lachaud et al. 1999; Rensing & Samuels 2004; Prislan et al. 2011). Several authors have reported that lipid droplets are present only in dormant cambium (Robards & Kidwai 1969; Rao & Dave 1983; Farrar & Evert 1997b; Prislan et al. 2011). Amyloplasts (starch-containing plastids), however, are numerous in the active state and absent or rare in dormant cambium, as observed in different species (Itoh 1971; Farrar & Evert 1997b; Begum et al. 2010a; Prislan et al. 2011). Begum et al. (2010a) pointed out that storage materials (e.g., lipid droplets and starch-containing plastids) are important for the dormancy and reactivation of cambium. During cambial dormancy, levels of starch might be low as a consequence of the breakdown of starch that is associated with the generation of energy for the development of cold hardiness (Pomeroy & Siminovitch 1971; Timell 1986). Lipid droplets might be utilised as sources of energy for cell division and the biosynthesis of new cell wall material in the cambium (Begum et al. 2010a). Seasonal changes in the ultrastructure of cambial cells may depend on the tree species and sites. Dictyosomes, for example, were found to be active in the dormant period in Aesculus hippocastanum (Barnett 1992) and Pinus strobus (Srivastava & O’Brien 1966). The appearance of different types of ER (tubular, vesicular or cisternal) in the cambium can also differ between active and dormant states (e.g., Rao & Dave 1983; Farrar & Evert 1997b) and among species (Srivastava & O’Brien 1966; Barnett 1992). These differences can be species specific or can be attributed to differences in growth conditions, different methodological approaches etc.

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Seasonal changes of cambial cells are mainly examined in transverse sections; however, ultrastructural features during the cytokinesis of fusiform cambial cells must also be studied in the radial plane. Bailey (1919), for example, described in detail the process of cell divisions in Pinus strobus based on observation with light microscope. Although periclinal divisions are most common in cambium, little is known about their ultrastructure (Rensing et al. 2002). In contrast to cells in primary meristems, the length of cambial cells can be 500 times larger than their diameter, so the course of divisions along the axis is slightly different (Rensing et al. 2002; Samuels et al. 2006). During cambial cell cytokinesis, mitotic spindles separate chromosomes across the radial width /dimension of the cells. However, during cell plate growth, the formed phragmoplast divides axially into two parts. Both parts are surrounded by cytoplasm, representing so-called boluses, which migrate in opposite directions along the extended axis of the cell and form the new cell plate (Rensing et al. 2002).

Figure 3. Schematic diagram of cytoplasmic changes occurring in cambial cells of temperate trees during a seasonal cycle. Actively dividing cell in spring or summer, with a large vacuole (V). Transition to rest in autumn; fragmentation of the vacuole (V) and thickening of the cell walls begins (W). Dormant cell in winter; numerous vacuoles become globular during the cessation of cyclosis; mostly smooth endoplasmic reticulum (ER), Golgi apparatus (G) with few secretory vesicles, numerous lipid droplets (LD). Transition to activity in late winter-early spring, showing elongation and fusion of vacuoles following the resumption of cyclosis, rough endoplasmic reticulum, active Golgi apparatus. Nucleus (N) with nucleolus (Nu), plasmodesma (pl). – (1–6) Micrographs showing transverse sections of Fagus sylvatica cambial cells in different stages of activity; (1, 2) dormant cells, (3) transition from dormant to active state, (4, 5) active cells and (6) transition from active to dormant state.

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Relatively little is known about the structural changes of the cambial cell wall during the transition from the dormant to the active state. In the majority of tree species, the walls of dormant cambial cells are thicker than those in active cells (Larson 1994). Chen et al. (2010) found that dormant cambial cell walls of Populus tomentosa displayed a multi-layered structure, denser fibril network, smaller pore size and fewer crosslinks between microfibrils than active cambial cell walls. Chaffey et al. (1998) suggested, based on observations of cambial seasonal changes in Aesculus hippocastanum, that cell-wall thickening at the onset of cambial dormancy should be considered to be secondary thickening and that selective lysis of this secondary wall layer during cambial reactivation restores the thinner, primary wall around active cambial cells. A certain seasonal variability in the frequency of plasmodesmata was also observed in Populus nigra (Fuchs et al. 2010). OBSERVATION OF CAMBIAL STRUCTURE WITH LIGHT (LM) AND TRANSMISSION ELECTRON MICROSCOPY (TEM)

Processing plant tissues for TEM can be divided into six major steps: (I) specimen acquisition from a living tree, (II) trimming of the specimen, (III) fixation, (IV) dehydration, (V) infiltration and (VI) embedding (Bozzola & Russell 1999). Standard techniques for wood formation studies are clearly outlined in “Wood formation in trees – cell and molecular biology techniques”, edited by Chaffey (2002); from light microscopy to advanced electron microscopy techniques. Recent advances in methodologies for studying the structure of cambium and developing xylem tissue are especially pronounced in the field of tissue fixation; Rensing et al. (2002) precisely presented differences in the observation of cambial cells when using conventional chemical fixation or cryofixation and substitution. We demonstrated that combining LM and conventional TEM can provide detailed information on cambial phenology and seasonal ultra-structural changes in cambial cells of Fagus sylvatica growing at forest sites with different weather conditions (Prislan et al. 2011). Sample collection In wood /phloem formation studies at cellular and ultrastructural levels, tissues containing phloem, cambium and outer xylem are collected from living trees. The time intervals of samplings should be relatively short, i.e., one to two weeks, and depend on the goal of the study. For observations of the seasonal dynamics of different phases of wood formation, small micro-cores are usually collected with tools causing minor damage on tree stems. A Trephor tool has recently become widely used for sampling (Rossi et al. 2006) but an increment puncher (Forster et al. 2000) and injection needles (Jyske et al. 2011) are also used. Due to the small size of wounds caused by these tools, repeated sampling on the same tree can be performed in more than one growing season, without affecting the vitality of the tree. However, micro-cores (e.g., diameter c. 2 mm and length c. 10 mm) are difficult to handle and they can be easily damaged or the tissue can be affected. They are therefore not suitable for ultrastructural observations, thus sampling larger blocks of intact tissues using a chisel and knife, as described by Uggla and Sundberg (2002) or Gričar et al. (2007b), should be preferred.

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Sample preparation and observation For light microscopic (LM) observations, microcores are first fixed in a solution, such as formalin-ethanol-acetic acid (FEA), dehydrated in a graded series of ethanol and clearing reagent (e.g. D-limonene) and infiltrated, as well as embedded in paraffin as described by Rossi et al. (2006). Various embedding media, such as glycolmethacrylate (Oberhuber & Gruber 2010) or polyethyleneglycol (Liang et al. 2009), are also used (Table 1). Cross sections of 8 to10 μm thickness are prepared with a rotary microtome and then stained with safranin and astra blue (e.g., Van der Werf et al. 2007; Gričar et al. 2007a), or cresyl violet acetate (e.g., Antonova & Stasova 1993; Deslauriers et al. 2003). For preparation of permanent sections, embedding media such as Euparal (e.g., Gričar et al. 2005) or Canada balsam (e.g., Moser et al. 2010) are used. Using LM, cambial phenology (onset and cessation of cambial cell production), phases of Table 1. Overview of sample processing procedures for different tissues and microscopy techniques. Light microscopy (LM) Use in wood formation studies Fixation

Dehydration

Transmission electron microscopy (TEM)

Cambial phenology, Seasonal changes in cambial ultrastrucxylem/phloem differentiation, ture (structure of living tissues should seasonal dynamics of growth be preserved). ring formation. Primary fixative: - Formalin-ethanol-acetic acid - Mixture of 5% glutaraldehyde, 8% solution (FEA) (Gričar et al. paraformaldehyde and 0.3 M cacody 2007b); late buffer) (Farrar & Evert 1997b; - Ethanol, propionic acid and Frankenstein et al. 2005); formaldehyde solution - Mixture of 2.5% glutaraldehyde in (Oberhuber & Gruber 2010); 0.05 M phosphate buffer (pH 6.8) - Water and ethanol solution (Rensing & Samuels 2004). (Lupi et al. 2010). Secondary fixative: (2% aqueous osmium tetroxide solution). Graded series of ethanol. Acetone. Clearing reagent: - D-limonene (Gričar et al. 2007a); - Histosol (Lupi et al. 2010).

Infiltration / embedding

Embedding media: - Paraffin (Rossi et al. 2006); - Glycolmethacrylate (Oberhuber & Gruber 2010); - Polyethyleneglycol (Liang et al. 2009).

Epoxy resin (Spurr 1969).

Cutting

Rotary microtome (sections 8–12 µm).

Ultra microtome (sections 90–100 nm).

Staining

- Safranin and astrablue (Gričar et al. 2007b); - Cresyl violet acetate (Deslauriers et al. (2003).

Uranyl acetate and lead citrate.

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differentiation of xylem /phloem cells and seasonal dynamics of xylem /phloem growth ring formation can be observed (e.g., Schmitt et al. 2000; Gričar 2007; Seo et al. 2008; Rossi et al. 2011; Michelot et al. 2012) (Table 1). Larger blocks of intact tissue are commonly collected for ultrastructural observations by transmission electron microscopy (TEM), because of easier manipulation and to prevent deformation of the tissue and the occurrence of artefacts. Afterwards, sample size is reduced to less than 2 mm in thickness in order to ensure adequate fixation (Bozzola & Russell 1999). Samples for observation of ultrastructural seasonal changes in cambial cells are fixed for one day in a mixture of 5% glutaraldehyde, 8% paraformaldehyde and 0.3 M cacodylate buffer. They are then washed in 0.1 M cacodylate buffer (pH 7.3) and post-fixed for one additional day in a 2% aqueous osmium tetroxide solution. They are again washed in 0.1 M cacodylate buffer (pH 7.3), dehydrated through a graded series of acetone and finally embedded in Spurr’s (1969) epoxy resin. Phosphate buffer is often used instead of cacodylate buffer (Rensing & Samuels 2004) (Table 1). Ultrathin transverse sections (90–100 nm) of cambium are then prepared. Sections are placed on copper grids and stained with uranyl acetate and lead citrate and examined with a TEM at an accelerating voltage of 80 or 100 kV (e.g., Farrar & Evert 1997b; Frankenstein et al. 2005; Prislan et al. 2011). Seasonal changes in the cytoplasm of cambial cells can be observed by TEM (Farrar & Evert 1997b; Rensing & Samuels 2004), as well as changes in the architecture of cambial cell walls (Chen et al. 2010). ENVIRONMENTAL REGULATION OF CAMBIAL ACTIVITY EXEMPLIFIED BY FAGUS SYLVATICA

We illustrate this section with our own examination of cambial phenology in Fagus sylvatica at two sites in Slovenia, central Europe, with different elevations and weather regimes. The low elevation forest site (400 m a.s.l.) has a mean annual temperature (MAT) of 11.3 °C and 1565 mm of annual precipitation and the high elevation site (1200 m a. s. l.) has 6.6 °C MAT and slightly higher precipitation (Prislan et al. 2011; 2013). The two sites were carefully selected based on previous investigations in beech and climatic factors in Slovenia involving tree-ring variation and climate (Di Filippo et al. 2007; Čufar et al. 2008), leaf phenology (Čufar et al. 2012), climatic situation and trends (De Luis et al. 2012), and connection of tree-ring variation, leaf phenology, cambial activity and wood formation and climate (Čufar et al. 2008; Prislan et al. 2013). Thus, the selected locations are representative for growth of beech at low and high elevations in Slovenia. Dormant cambium contained 4 to 5 cell layers at both sites, whereas active cambium had a slightly higher number of cell layers at the low elevation site (around 11) than at the high elevation site (around 8) (Prislan et al. 2011). At the high elevation site, the onset of cambial cell production occurred one month later (in the middle of May) than at the low elevation site (Prislan et al. 2013). Maximal cell productivity was observed at the high elevation site around the summer solstice and at the low elevation site at the beginning of June. Cell production ceased at the beginning of August at the high elevation site and at the end of August at the low elevation site (Fig. 4) (Prislan et al. 2011).

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Figure 4. Light micrographs showing the cambium of Fagus sylvatica (a) in the dormant state before the onset and (c) after the cessation of cambial activity and (b) active cambium. The graph shows seasonal changes in the number of cells in the cambium at high and low elevation sites (d). Arrowheads indicate the timing of maximal cambial productivity; at the high elevation site this occurred, around the summer solstice, at the low elevation site in the beginning of June. Arrows show the duration of cambial cell production.

Our study demonstrated that the duration of the growing season is inversely proportional to the altitude (in temperate regions); the onset of cell production at a high elevation starts later and ceases earlier (Prislan et al. 2013). However, this conclusion is preliminary and for its generalisation about the effect of elevation on cambial phenology further studies should be performed with the inclusion of additional sites. The shorter duration of cambial activity at the higher sites can be ascribed to the generally lower air and soil temperatures, as well as a longer period of snow cover, since such conditions are limiting for physiological processes, particularly at the beginning of the vegetation period, as reported for instance by Kirdyanov et al. (2003) and Moser et al. (2010).

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The cambium is mainly susceptible to environmental signals during the active period and archives them in the wood and bark structure (e.g., Frankenstein et al. 2005; Gričar & Čufar 2008). In temperate and cool regions, temperature and photoperiod are important external factors of the initiation of cambial reactivation and xylem differentiation (Li et al. 2009; Begum et al. 2013). Deslauriers et al. (2008) studied cambial phenology in Pinus leucodermis at high altitudes in Italy and showed that temperatures in spring are the main factor driving the onset of cell production. Rossi et al. (2008) tried to define temperature thresholds for the onset and cessation of cambial cell production for conifers from different latitudes of boreal and temperate regions. The presented mean critical temperatures, which varied by around 8 °C, were significant for the onset of cell production; however, mean critical temperatures for cessation (around 14 °C) were not significant. Seo et al. (2011) monitored the intra-annual growth dynamics of pine trees (Pinus sylvestris) in northern Scandinavia, with diverging results; at some sites, wood formation was mainly positively correlated with temperature, whereas such a positive correlation was missing at other sites or it was even negative. Furthermore, we also showed that the temperatures prior to the occurrence of relevant cambial phenological phases, together with calculated growing degree days, significantly differed at the low and high elevation beech sites in Slovenia, indicating that phenological events are not in simple relationship with climate, or at least not in agreement with year-to-year variations in weather. The accumulated heat units (growing degree days - GGDs) at the beginning of cambial activity, for instance, were higher in the lowland (around 147 °D) than at higher elevations (around 72 °D) (Prislan et al. 2013). Seasonal changes in the ultrastructure of cambial cells studied along the altitudinal gradient are rare. We showed by TEM that processes in cambial cells that are related to seasonal changes are similar regardless of the elevation; only the timings of individual events differ and are generally delayed at higher elevations at the onset of the growing season. At the end of the growing season, the sequence of changes in the cambium was just the opposite; the transition from active to dormant state started first in the cambial cells of Fagus sylvatica from the higher elevation (Prislan et al. 2011). LM and TEM, complementary approaches to studying cambium seasonality

This section is also illustrated by our own results obtained by a combination of TEM and LM studies of Fagus sylvatica from two elevations in Slovenia. TEM observations revealed that cytoplasmic changes in cambial cells during the seasonal cycle occur much earlier (about one month) than was observed at the cellular level with LM (Prislan et al. 2011). The results are affected by: 1) the media used for tissue fixation and embedding, as well as 2) the thickness of sections and microscope resolution/magnification. A fixative such as FAA (formaldehyde, ethanol and acetic acid) is commonly used in wood formation studies (observing cambial phenology and xylem growth ring formation

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dynamic), whereby sections are observed with LM. The main disadvantage of FAA is insufficient preservation of the cytoplasm in cambial cells. Reactivation of the cambium at the cellular level is usually histologically defined by an increased number of cambial cells and the occurrence of newly formed xylem and phloem cells in early developmental stages. With LM, it is possible to detect cambial production based on the number of newly formed cells at a certain time interval, as well as differentiation of xylem and phloem cells; however, cytoplasmic changes associated with the seasonal cycle of the cambium cannot be recognised (Prislan et al. 2011). Observations on a sub-cellular level are also possible with LM (Fig. 5), when using primary fixation with glutaraldehyde and phosphate buffer and secondary fixation in osmium tetroxide (usually used for TEM observations), as demonstrated by Bailey (1919) or Begum et al. (2010b), who were able to observe new cell plates. Similarly, Rensing and Samuels (2004) were able to observe differences in the arrangement of vacuoles between dormant and active cambial cells. TEM, with its high resolution and magnification (Goodhew et al. 2000), in combination with proper fixation of the cambial tissue, i.e., preservation of cytoplasm, allows the observation of seasonal changes in the distribution and size of cell organelles (Fig. 5). This is particularly important during the transition of cambial cells to active or dormant state, when the number of cell layers is unchanged but ultra-structural changes (in the form of dictyosome activity, changes related to endoplasmic reticulum, etc.) are already occurring. Consequently, different criteria for cambial reactivation are used with different sample preparation and microscopy techniques (resolutions), as was already stressed by Frankenstein et al. (2005). Begum et al. (2013), e.g., called the period from late winter to late spring, when new cell plates are formed in the cambium, “cambial reactivation”. In our TEM study (Prislan et al. 2011), changes in the ultrastructure of cambial cells were observed prior to the formation of new cell walls, and the cambium can thus be considered to be active as well in this earlier stage. When using different fixation, embedding and microscopy techniques and criteria, the established dates of cambial seasonality can vary. However, a combination of different approaches enables the activity and productivity of cambium to be precisely followed. CONCLUSIONS

Examination of cambial tissue using different microscopic levels revealed that different criteria are used to define the onset of cambial reactivation; the results of different studies are therefore not simply comparable. The terminology should thus be adjusted and standardised to avoid disagreement among different research groups when comparing data, as was already stressed by Frankenstein et al. (2005). Complementary methodologies can assure a better understanding of relations between physiological processes in the cambium and climate with regard to predictions of tree responses to anticipated climate change. The presented facts can help in future cambial studies, particularly when the results of different studies using different techniques must be compared.

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This work was supported by the Slovenian Research Agency, young researchers’ program (Peter Prislan) programs P4-0015 and P4-0107, and by the LLP ERASMUS bilateral agreement between the University of Ljubljana and the University of Hamburg. The cooperation among international partners was supported by the COST Action FP1106, STReESS. The authors gratefully acknowledge the help of Professor Jasna Štrus and her team at the Department of Biology, Biotechnical Faculty, University of Ljubljana. We thank Tanja Potsch, Dr. Magda Tušek Žnidarič and Dr. Nada Žnidaršič for their immense help in the laboratory.

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IAWAIAWA Journal 34 (4), 408–424 Journal 342013: (4), 2013

Visualizing wood anatomy in three dimensions with high-resolution X-ray micro-tomography (µCT) – a review – Craig R. Brodersen Horticultural Sciences Department, Citrus Research & Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, U. S. A. E-mail: [email protected]

Abstract

High-resolution X-ray micro-tomography (μCT) has emerged as one of the most promising new tools available to wood anatomists to study the three-dimensional organization of xylem networks. This non-destructive method faithfully reproduces the spatial relationships between the different cell types and allows the user to explore wood anatomy in new and innovative ways. With μCT imaging, the sample can be visualized in any plane and is not limited to a single section or exposed plane. Conventional CT software aids in the visualization of wood structures, and newly developed custom software can be used to rapidly automate the data extraction process, thereby accelerating the rate at which samples can be analyzed for research. In this review the origins of xylem reconstructions using traditional methods are discussed, as well as the current applications of μCT in plant biology and an overview of pertinent technical considerations associated with this technique. μCT imaging offers a new perspective on wood anatomy and highlights the importance of the relationships between wood structure and function. Keywords: Synchrotron, 3D, tomography, wood anatomy, visualization, μCT. Introduction

Over the past 10–15 years high resolution X-ray micro-computed tomography (μCT) has seen a surge in popularity as a tool for producing three-dimensional (3D) visualizations of plant tissue. This non-destructive method allows wood anatomists to repeatedly section a single block of wood from any perspective, selectively isolate a region of interest, and then repeat the same process ad infinitum throughout the sample with each iteration of this process digitally preserved. μCT technology has progressed to the point where it now rivals low magnification scanning electron microscopy (SEM) in its ability to resolve fine details. However, SEM can only visualize an exposed surface while μCT can be used to probe the entire sample and virtually expose a plane in any orientation. As this technology continues to mature, image resolution, quality, and acquisition time will improve and μCT has the potential to serve as an important tool in the analysis of wood structure and function. Increased interest in this new technique © International Association of Wood Anatomists, 2013 Published by Koninklijke Brill NV, Leiden

DOI 10.1163/22941932-00000033

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is indicative of the recognized importance of xylem network connectivity by wood anatomists and the inherent difficulty of visualizing the spatial organization of xylem networks in three dimensions. Studying the spatial organization of xylem vessels is challenging not only because of the scale at which the networks exist, but also because of the tools traditionally available to visualize such networks. Light microscopy is the most common visualization method, which applies a two-dimensional tool to a three-dimensional problem. In many species, vessels do not follow a straight course through the wood, but instead “drift” laterally around the stem, often resulting in spiral grain (Zimmermann & Brown 1971; André 2005). As a consequence, aligning a microtome to cut a radial or tangential plane through multiple vessels to track their ascent through a large sample of wood is exceedingly difficult, and a single transverse section reveals little about the axial course of a vessel or vessel group. Serial sectioning, therefore, has been the most popular method for reconstructing xylem networks (e. g. Burggraaf 1972; Bosshard & Kučera 1973), continues to be used regularly (Fujii et al. 2001; Kitin et al. 2004), and is one of the fundamental components of confocal microscopy (Kitin et al. 2003). From those sections the path or course of individual xylem conduits can be tracked through a length of plant tissue such that the connections between vessels can be determined. This exercise, while time consuming and at times tedious, is highly recommended for anyone interested in studying the spatial organization of the xylem, as it is one of the easiest ways to develop a three-dimensional understanding of the spatial relationships between different plant tissues. The serial sections and reconstructed xylem network reveal that vessels are not merely straight, vertical pipes, but dynamic structures that move or “drift” radially or tangentially around the stem. Serial sectioning also allows the viewer to study the elegant solutions plants have developed for the distribution of water through the bifurcation of vascular bundles that lead to leaf traces (McCulloh et al. 2003) or the rich diversity in stelar organization (Beck et al. 1982; Pittermann et al. 2013), often revealing relationships between structure and function of the xylem network that are otherwise obscured by the complexity of plant tissue (Brodersen et al. 2012). The development of the optical shuttle method was one of the key advancements in the reconstruction of xylem networks using serial sections (Zimmermann & Tomlinson 1966). The system developed by Zimmermann and Tomlinson (1966) featured a 16 mm film camera mounted to a microscope focused on the exposed transverse face of a stem sample mounted in a microtome. Following the removal of each transverse section a photograph was taken, and the serial images were then assembled into a film. Each frame could then be viewed sequentially in forward or reverse to scroll axially along the stem, where the z-axis of the stem was translated into time (e.g. Zimmermann & Brown 1971; Zimmermann & Tomlinson 1968). This method significantly decreased the time necessary to process a stem sample and preserved the sections as photographic images that could be reconstructed at a later time. However, reconstructing the network was performed manually by projecting the film onto tracing paper where the position of individual vessels could be tracked through a length of stem (Zimmermann & Brown 1971; Fig. 1). The resulting reconstructions revealed the complex and convoluted nature

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Figure 1. Partial reconstruction of the vessel network of Cedrela fissilis redrawn from Zimmermann and Brown (1971). Serial light micrographs were used to plot the course of vessels through a block of wood visualized using the optical shuttle method. Each vessel is numbered at the end where it enters or leaves the block of wood. The y-axis was foreshortened ten times.

of the vessel networks in many species. The method has been updated to couple serial sectioning with digital micro-photography, yielding excellent results (Kitin et al. 2004; André 2005; Huggett & Tomlinson 2010; Wu et al. 2011). The original films are still of use today because of the high quality of the serial sections, many of which have been digitized. As an example, one such film (Zimmermann 1971) was loaded into freely available software (Quicktime 7.0.1, Apple Computer Inc.; FIJI image processing software (a Java-based distribution of ImageJ)), and the individual frames of the film were extracted as an image sequence. Because of the known frame rate in the digital version, the original frame rate of the film version, and the section thickness, the image sequence was easily transformed into a 3D volume rendering using commercially available software (Avizo 7.0, VSG) (Fig. 2). The whole block of wood can be reconstructed from the image sequence (Fig. 2b), and individual vessels can be selected, reproduced as volume renderings, and viewed from a variety of angles to study the course of the vessels through the wood (Fig. 2c, d). Much like the original films, the axial scale can be compressed (Fig. 1, 2b–d), or displayed to show the true scale of the sample (Fig. 2e). While this example shows that films produced some 40 years ago can be quickly reconstructed using modern computer software, it should be noted that this method is easily adaptable to serial sections created today. While the optical shuttle equipment makes the process much faster, traditional serial sections can be used with the same software to make reconstructions of xylem networks. Currently, μCT systems often have limited access, and digital serial sections created using light microscopy could be a simple and inexpensive alternative if imaging in a single plane is sufficient.

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Figure 2. Three-dimensional reconstruction of Cedrela odorata xylem vessels using visualization software. Transverse serial images of the wood (a) were extracted from the film produced by Zimmermann and Brown (1971) by isolating individual frames in the film and then loading them into 3D visualization software. The block of wood could then be visualized as a whole (b), foreshortened in the y-axis as originally presented by Zimmermann and Brown (1971), as selected vessel groups (c, d), or fully expanded in the y-axis (e). The software readily allows the user to visualize the network from different perspectives (c, d) to track the course of the vessels through the wood. The long edge of the wood block is X µm, and scale varies with perspective in b–e. In (e) the length of the block is X µm.

Technical considerations for visualizing wood anatomy with µCT imaging

Plant research utilizing μCT has proliferated over the past decade and these studies were facilitated by the presence of lab-based μCT systems in university imaging facili-

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ties and the development of synchrotron-based microtomography instruments at a limited number of facilities around the world (e.g. the Lawrence Berkeley National Laboratory Advanced Light Source (USA), Swiss Light Source (Switzerland), Australian Synchrotron, etc.). Lab-based systems now offer comparable image resolution compared to synchrotron instruments, but the primary advantage of synchrotron μCT is the high flux that allows for shorter exposure times, and therefore shorter overall scan times, and a wider range of available X-ray energies. While μCT is not the only non-destructive method for visualizing plant tissue (Bucur 2003), the advantages of using μCT for studying wood anatomy is becoming clear. Magnetic resonance imaging is an alternative to μCT for 3D imaging, and Oven et al. (2011) have shown the utility of this tool for studying plant tissue at a lower resolution than μCT, but free of any issues related to X-ray exposure. The μCT instruments used for studying plants are based on the same principles as medical CT systems. Throughout the literature the method has been given many names and the field has yet to settle on a specific acronym (e. g. micro computed tomography (μCT), high-resolution computed tomography (HRCT), high-resolution X-ray computed tomography (HRXCT), X-ray micro computed tomography (XMCT), etc.); however, they all refer to the same method with slight variations depending on the X-ray source and facility configuration. Briefly, X-rays aimed at a sample are attenuated based on the absorption properties of the sample’s constituents. Opposite the X-ray source is a scintillator that converts X-rays into visible light, which is then directed with a series of lenses and mirrors to a CCD camera that captures a single projection image. The current CCD camera utilized by the Advanced Light Source in Berkeley, CA has a 4008 × 2672 pixel array, and at the 4.5 μm resolution the field of view is 18 × 12 mm. The sample is then rotated in small increments (e.g. 0.125°) over 180°, with a projection image taken after each angular increment. These projection images are then normalized for image intensity, background corrected, and then “reconstructed” into a set of digital serial images composed of voxels (volumetric pixel elements) instead of pixels. Each voxel is assigned an x, y, and z coordinate and an intensity value corresponding to X-ray attenuation for that point in three-dimensional space. The result is a stack of digital images not unlike a set of serial light micrographs. Contrast in μCT imaging is achieved through the natural attenuation of X-rays by the sample. Regions within the sample that absorb fewer X-rays appear as dark voxels, and dense areas appear white or light gray voxels. Cellulose and other carbon-based compounds readily absorb X-rays, and the difference in X-ray attenuation between plant tissue and the surrounding air provides excellent contrast. Contrast agents can be injected into xylem vessels (e.g. KI solutions or silicone resin, see Brodersen et al. 2011), but most conventional (i.e. medical) contrast agents are incompatible with live plant tissue or readily diffuse through the porous cell walls of dry plant tissue. Many image visualization software packages are available for visualizing μCT datasets, including both commercial (Avizo (VSG, Inc., Burlington, Massachusetts, USA); VGStudioMax (Volume Graphics GmbH, Heidelberg, Germany), etc.) and free options (Fiji (www. fiji.sc/Fiji); Drishti (anusf.anu.edu.au/Vizlab/drishti)). In addition, several wood anatomy research

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groups have developed custom software packages to automate the analysis of wood μCT images (e.g. Steppe et al. 2004; Brodersen et al. 2011). Dried plant tissue is particularly amenable to μCT imaging because of the relatively large difference in X-ray attenuation between plant tissue and air inside the vessel or tracheid lumen. Small diameter cells (e.g. < 20 μm) are less easily visualized at lower resolution (e.g. > 5 μm) and benefit from high resolution scans. In live plant tissue or excised fresh tissue that remains hydrated, the air-filled xylem conduits are easily distinguished, while the surrounding water-filled fibers and parenchyma are difficult to visualize. However, phase contrast μCT can help to enhance the outline of the cell walls, but contrast in the voxel intensity between the cellular water and the cell walls is often insufficient to separate the two substances during image segmentation (Brodersen et al. 2011). Recently, Blonder et al. (2012) used μCT (lab-based and synchrotron) to show leaf venation could be imaged using this technique, providing an alternative to traditional methods for clearing leaves. As noted above, the resolving power of μCT instruments continues to improve, and many facilities allow imaging at three to four different magnifications. However, increasing the resolution decreases the field of view, not unlike traditional light microscopy optics, and resolution should be selected based on the requirements of the investigation. For example, a xylem network composed of vessels approximately 20 μm in diameter scanned with a 5 μm resolution would yield an image with four voxels that span the vessel lumen. In most μCT systems some image noise is inevitable, and low signal:noise can lead to images with low contrast between the vessel lumen and the surrounding plant tissue. With such a low sampling of voxels inside the lumen visualizing the important details (e.g. connections, pitting, vessel endings) can be difficult. One solution is to increase resolution at the expense of a smaller field of view. In our example, decreasing the voxel size to 2 μm or smaller effectively doubles the number of voxels representing the vessel lumen. Most μCT systems allow for “tiling”, where the sample can be shifted in the X-ray path such that subsequent scans can seamlessly capture an adjoining area of the sample with only 5–10 μm of overlap required between scans for registration. Using image-processing software these tiles can be combined to create a continuous dataset. This technique can be useful for scanning at high resolution to study xylem organization outside of the field of view of a single scan, or allow the user to scan long sections of plant material. The implementation of larger image sensors will ameliorate some of these issues in the future and reduce the overall number of tiles necessary to capture an entire xylem network. Low resolution scans are usually sufficient to determine xylem conduit connectivity in (i.e. < 1 cm in diameter) provided the vessel diameters are large (e.g. 4.5 μm resolution used by Brodersen et al. 2012, 2013 to study grapevine vessels ~75–200 μm in diameter), while higher resolution imaging can reveal fine details like the location of intervessel wall pitting (e.g. Trtik et al. 2007; Van den Bulcke et al. 2008). Figure 3 shows the xylem network from the petiole of Citrus sinensis at 650 nm resolution. Because of the high resolution, both vessel-parenchyma and intervessel scalariform pitting are visible as well as the annulus delineating individual vessel elements (Fig. 3b, c). In a recent paper, Mayo et al. (2010) present images of the same wood sample visualized at three different resolutions and clearly show the advantages of each.

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Figure 3. Three-dimensional volume rendering of a Citrus sinensis petiole visualized with µCT imaging (a) performed at the LBNL Advanced Light Source in Berkeley, CA, U.S.A. (Beamline 8.3.2). When visualized at 600 nm resolution, vessel wall details are visible including scalariform pitting and the annulus delineating vessel elements (white arrow). Calcium oxalate crystals are also visible (black arrow) embedded in the cortex. Panel (b) reveals a higher magnification region of (a). Scale varies with perspective, but the cylinder of tissue visualized in (a) is 1.7 mm in diameter, and the vessels in (b) are 20 µm in diameter. In (c), scalariform intervessel pitting is clearly visible (black arrow) as well as vessel-parenchyma pitting (white arrow).

Examples of µCT imaging applications for wood anatomists

For wood anatomists, μCT has proven to be a highly useful tool that will aid in answering questions about the three-dimensional organization of the xylem and its relationship to the surrounding tissues. High resolution imaging systems now provide sufficient resolving power to clearly visualize the xylem in species bearing tracheids or small-diameter vessels and capturing the fine details of the conduit walls that are otherwise obscured with lower resolution scans (e.g. Fig. 3; Mayo et al. 2010). Of equal importance is the simplicity by which wood can be virtually dissected using μCT, and this technology will help wood anatomists to better understand the spatial arrangement of the xylem conduits and the supporting tissues. Recent research has shown that paratracheal parenchyma cells are of particular importance in the dynamic process of drought

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recovery (Tyree et al. 1999; Salleo et al. 2004; Brodersen et al. 2010; Brodersen & McElrone 2013), and μCT has proven to be an important tool in understanding this relationship. While we are endowed with a thorough knowledge of wood anatomy owing to the rich history of histologic wood preparations visualized with light microscopy and other techniques (Schweingruber 1990, 2011; Carlquist 2001; Tyree & Zimmermann 2002; Evert 2006), the three-dimensional relationships between tissue types in wood remain difficult to visualize with traditional techniques. μCT offers an additional, complementary tool to traditional methods. For example, Robert et al. (2011) utilized μCT imaging to study the network structure of the xylem and phloem in Avicennia wood, revealing complex patterns of concentric, successive cambia. Such studies will help reveal the relationships between the structure and function of the xylem, particularly when framed in an ecological context. In another example, Page et al. (2011) used μCT to study the 3D organization of the xylem in co-occurring Acacia species from Australia. In this study the authors found that the degree of xylem connectivity was similar in all three species despite differences in branch water potential and other anatomical traits. Drought tolerance may be more tightly correlated with leaf shape than xylem anatomy within the leaf, providing yet another example of how selection can act at different levels of organization. Explorations into the 3D organization of the xylem show the future potential of μCT as a transformative tool that may further expand or redefine the wood anatomist’s nomenclature as physiologically important structures become apparent. One such example is the diversity of traits related to vessel grouping and how to determine from cross sections which vessels are connected through a shared wall (Carlquist 1984). Hass et al. (2010) used μCT to study the porosity of beech wood (presumably Fagus sylvatica) and identified groups (“clusters”) of vessels that appeared to be connected within the block of wood. Connectivity was assumed when the vessels were in close enough proximity to each other or the two vessels appeared to merge. As the image resolution in the Hass et al. 2010 study was insufficient to visualize intervessel pitting, the problem arises of when to characterize vessels as being connected and whether vessels within a group are connected and over what axial length. One can imagine viewing a transverse section where vessels are grouped in pairs or triplets. Serial sectioning might reveal that those pairs exist for only a short axial distance and the connection is fleeting. Or, upon closer inspection, the group is within close proximity but not connected (Brodersen et al. 2011). With sufficient sampling vessel grouping characteristics can be obtained with confidence, and the total shared wall area and contact length between vessels can be approximated with serial light microscopy (Wheeler et al. 2005), a value that may be the much more important for water transport or drought resistance than the number of vessels in a group. The two characteristics are undoubtedly related, but as the 3D course of xylem conduits becomes better characterized in the future, wood anatomists will need to settle on a new set of characteristics with which to describe these three-dimensional relationships. For example, how should we define the degree of contact between the xylem conduits and the ray parenchyma, particularly when we consider the organization in 3D? Here, the stem of a Pinus taeda seedling was scanned and visualized with μCT

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Figure 4. Three-dimensional µCT reconstruction of the stem of a Pinus taeda seedling scanned at 650 nm resolution. The volumetric rendering of the stem, approximately 1.2 mm in diameter was visualized as a whole (a), and was then virtually dissected to expose the a longitudinal plane (b) to expose some of the tracheids and the rays (arrow). In an alternate orientation, the rays and tracheids from a region of the sample were reconstructed from the 3D dataset (c). Once extracted, the 3D volumes can then be viewed from a wide range of perspectives (d, e, f) to reveal the spatial relationships between the two cell types. Scale varies with perspective.

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imaging (Fig. 4). The 3D volume of the stem was visualized (Fig. 4a), and then dissected to expose a longitudinal plane through the wood revealing the tracheids and rays (Fig. 4b). Using computer software the tracheid network was reconstructed as well as the rays embedded in the xylem (Fig. 4c). Measuring the precise amount of ray-tracheid overlap with traditional light microscopy would be extremely difficult as the amount of overlap changes axially through the stem. Using μCT the images presented in Figure 4 were assembled in less than an hour using Octopus 8.3 (Institute for Nuclear Sciences, University of Ghent, Belgium) for the image reconstruction and Avizo 7.0 (VSG, Inc., Burlington, Massachusetts, USA) software for visualization (Brodersen et al. 2011), with an additional 45 minutes for scanning and mounting the sample. This method allows the ray-tracheid network to be visualized from a variety of different angles (Fig. 4d–f) and measuring the connectivity of the network is easily managed. Figure 4 represents a stem that is less than 1 mm in diameter and length, and reveals only a small fraction of the rays present in the sample. How much variability exists within the sample, throughout the whole stem, or between individual plants? By tiling several μCT scans together to capture a longer section of the stem and then broadly sampling from a population one could attempt to answer these questions. At this early stage of our inquiries into 3D xylem organization using μCT it will be important to identify the relevant traditional characters (Wheeler et al. 1989) and whether they can continue to be informative when considering both 2D and 3D applications. Ideally, each characteristic wood anatomists use to characterize a wood specimen would be valid both in 2D and 3D. The vast majority of anatomical characters will remain valid, but the degree of connectivity between vessels, and other cell types, may necessitate a new set of descriptive terms to document these anatomical traits in 3D. Live plant imaging

In addition to imaging dehydrated samples, intact plants can be visualized in vivo with μCT without compromising the tension on the xylem sap. This method has been used to study the spread of drought-induced embolism (Brodersen et al. 2013b) as well as the mechanism responsible for removing embolisms from the xylem network (Brodersen et al. 2010; Suuronen et al. 2013). While in vivo μCT imaging is still new, this method holds great promise for studying the functional status of intact xylem networks at resolutions that are significantly better than other non-destructive imaging tools (e.g. nuclear magnetic resonance (NMR) imaging; Holbrook et al. 2001). Studies specifically focused on visualizing the functional status of the xylem in vivo have broadened the utility of μCT and continue to emphasize the link between form and function. Currently, μCT and NMR are the two best methods available for monitoring the functional status of the xylem in vivo. One of the biggest technological obstacles for future μCT studies in live plants is the dependence of image contrast on the X-ray attenuation differences between air and water. In hydrated tissue, the vessel lumen is easily visualized when filled with air, but the surrounding, water-filled tissue is largely obscured thereby making it difficult to distinguish the anatomy of the surrounding tissue (Brodersen et al. 2011). μCT images

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can be coupled with brightfield light microscopy or SEM images of the tissue following μCT scanning to get a comprehensive understanding of the spatial organization of the xylem. Finally, μCT has been used in combination with other methods for determining physiological parameters such as cavitation resistance, and allows for a visual confirmation of the spread of embolism that is otherwise measured using indirect methods (e.g. Choat et al. 2010; McElrone et al. 2012). Other plant related applications

μCT imaging has not been limited to the wood structure alone and has been employed in a wide range of applications related to plant biology. Dhondt et al. (2010) used μCT to study the morphological traits of Arabidopsis thaliana seedlings, including the trichomes on the leaf surface and detailed imaging of flower parts. Kaminuma et al. (2008) expanded on this line of research and used μCT to study the 3D anatomy of trichomes and their distribution on the surface of A. thaliana leaves, linking specific genes to the distribution of the trichomes on the leaf lamina, providing another method for phenotyping A. thaliana mutants. Low-resolution imaging can also be useful for studying the gross morphology of whole plant structures, such as the graft unions in stems (Milien et al. 2012), the anatomy of flower parts (Stuppy et al. 2003), or whole pieces of fruit (Verboven et al. 2008). Brodersen et al. (2012) used μCT to study the organization of the vascular bundles in two fern species, and by combining those images with ecophysiological tools were able to better understand the fundamental relationships between the structure and function of the xylem at a higher organizational level. This technology has also been used successfully in the field of dendrochronology (e.g. Okochi et al. 2007; Grabner et al. 2009; Bill et al. 2012), with newer μCT instruments being capable of distinguishing between latewood and earlywood in tree rings. The ease of use and prevalence of traditional tree coring technology, however, appears to supersede the widespread use of μCT; however, automated image analysis and the three-dimensional capabilities could reveal additional characteristics that might be important to that field (Fonti et al. 2010). X-ray techniques have also found their way into the field of paleobotany (Boyce et al. 2003; DeVore et al. 2006; Smith et al. 2009; Scott et al. 2009), and due to the difficult nature of mineralized wood samples, μCT imaging may make previously intractable samples more amenable to research. Recent advancements and combinations of different types of technology with μCT are promising and have shown the utility of this method for wood analysis from an industry perspective. For example, De Vetter et al. (2006) paired μCT with SEM X-ray spectroscopy to study cell wall penetration of chemical wood additives, Panthapulakkal and Sain (2013) studied changes in wood structure due to a chemical treatment, Taylor et al. (2013) used μCT to study wood shrinkage following dehydration, and Derome et al. (2011) found that μCT could be used to determine differences between earlywood and latewood shrinking and swelling in Picea abies wood samples. μCT technology is also being applied at the intersection of food science and wood anatomy, where Porter et al. (2011) used μCT to study the porosity of wood from different types of wine barrels. Forsberg et al. 2008 used μCT to study strain and wood deformation resulting

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in 3D displacement field diagrams. X-ray scattering and μCT were recently used to determine the differences in the microfibril orientations in the conduit walls of three different tree species (Svendström et al. 2012). The technology also has the potential to significantly improve the mathematical models that have previously been based on xylem parameters picked from the literature to simulate a xylem network (e.g. Loepfe et al. 2007). By scanning entire vessel networks with μCT and then importing those three-dimensional data to populate the network models, water transport through the real xylem network can be simulated, including the response of the network to dysfunction resulting from drought-induced embolism, tylose formation, or the introduction of pathogens. Using this method, Lee et al. (2013) found that the inclusion or exclusion of xylem vessel relays (Brodersen et al. 2013a) from the network significantly impacts the redundancy of water transport, and under certain circumstances can generate scenarios where reverse (i.e. basipetal) flow is predicted. The identification of the spatial distribution of intervessel pitting should allow for more sophisticated modeling of water transport throughout xylem networks, and results from these simulations will be realistic and true to the original plant sample. Technical considerations and potential pitfalls

Selecting an appropriate image resolution is of critical importance, as many of the current bottlenecks associated with μCT imaging are related to the large size of the image datasets. Resolution scales proportionally to the final dataset size, as the higher number of pixels in high-resolution images result in larger file sizes. Because the field of view decreases with increasing resolution, tiling is often required to visualize the whole sample. The datasets resulting from large tiling efforts can be substantial. For example, scanning a stem 8 mm in diameter over 5 mm imaged at 4.5 μm resolution following the methods of Brodersen et al. (2011) yields a stack of 1112 images, with a total size of 4–5 GB. The geometric scaling of data resulting from merging multiple tiles is sobering, particularly when considering the computer processing power, data storage, and graphics processing required to visualize the dataset as a whole. This tiling technique has been used to scan stem segments up to 4.5 cm in length (Brodersen et al. 2013a), but could be expanded to scan very long or wide segments. Theoretically, a tree trunk several centimeters in diameter could be scanned, with the resulting 3D image composed of hundreds of tiles. Each μCT instrument is different, and the dimensions of the sample stage and its range of motion will be the primary limitation to acquiring the data. Once the data is collected, the formidable task remains of merging the tiles into a continuous dataset and displaying it properly for analysis. At the time of writing, computers capable of handling such a dataset are exceedingly rare. However, computing power and data storage have decreased in cost during the past 10–15 years, but how long that industry can sustain this trend and follow Moore’s law is uncertain (Schaller 1997; Mack 2011). The images presented here were created using a custom-built computer with 24 processors, 96 GB of RAM, and a dedicated high-end graphics processor. These computer systems are expensive,

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but costs are progressively decreasing and universities often have computers that are associated with the lab-based μCT systems. A significant bottleneck in the μCT technique is image analysis. A variety of visualization software packages are available, both commercial and open-source, each with their own merits and the type of research will dictate the selection of an appropriate software package. This technology generates thousands of images, and developing a strategy to efficiently analyze the data is a significant challenge. Steppe et al. (2004) developed a custom computer program to automate μCT image analysis to measure vessel diameter, cross-sectional area of the xylem vessels, wood density, and other parameters. Similarly, Brodersen et al. (2011) expanded on that technique to automate the analysis of xylem anatomy and network connectivity. While the image preparation time was slightly more labor intensive using the automated software package (1 vs. 4 hours), the total amount of time required to analyze the same network information was significantly decreased (16 vs. 0.03 hours). The time was largely devoted to image processing which is dictated by computer processing power. Faster computers and new iterations of the software will accelerate this process, further reducing the amount of time necessary to analyze large datasets. Advances in image processing (e.g. Gil et al. 2009) will also improve the overall quality of the images prior to analysis with the aim of reducing image noise while preserving the inherent structures in the images. Conclusions

As both the temporal and spatial resolution improve in future iterations of both lab-based and synchrotron μCT systems image quality will continue to improve. By far the most important key to future use of this technology is widespread access and collaborative research. Currently, lab-based systems are expensive and synchrotron-based systems have limited access. Focused, well-defined, hypothesis-driven studies that bring together wood anatomists and physiologists are likely to yield the most significant results with the limited resources available. Future studies should focus on obtaining images of larger samples, multiple samples from the same species or several species within a genus, and determining variability of functional wood characteristics across a broad range of species. Pairing anatomical analysis with physiological measurements will help to strengthen our understanding of the link between form and function. As noted above, readily available user-friendly software that aids in the automation of xylem network analysis will make the method much more attractive to users unfamiliar with the technique and help to standardize the measurements so that comparative studies are possible. Finally, the highly visual nature of this technique has obvious implications for teaching plant anatomy. The 3D representations of xylem organization make the complexity of xylem networks less intimidating and it is easy to see the spatial relationships between the different cell types (e.g. Fig. 4). Instead of a two-dimensional image on a page, μCT brings the structures to life by giving them volume and form. Many of the 3D visualization software packages also allow components of the 3D models to be viewed as an interactive website with a typical student-level computer. Three-dimensional

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μCT images could also add a new facet to online wood databases such as InsideWood (insidewood.lib.ncsu.edu) and The Xylem Database (www.wsl.ch/dendro/xylemdb). Acknowledgements The author kindly thanks A. MacDowell and D. Parkinson for their assistance at the Lawrence Berkeley National Laboratory Advanced Light Source, Beamline 8.3.2 in Berkeley, CA, USA where the μCT imaging for this manuscript was performed. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Services, of the US Department of Energy under contract no. DE-AC01-05CH11231. Dr. D. Johnson kindly provided the Pinus taeda seedling for μCT imaging. The author also thanks C. Manuck, M. Reed, and A. McElrone for their thoughtful edits to this manuscript.

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Wegner IAWA et al. Journal – Vessels 34 (4), in diffuse-porous 2013: 425– 432 species

ROXAS – an efficient and accurate tool to detect vessels in diffuse-porous species Lena Wegner 1,*, Georg von Arx 2, Ute Sass-Klaassen1 and Britta Eilmann1 1 Forest

Ecology and Forest Management Group, Centre for Ecosystem Studies, Wageningen University, PO Box 47, 6700 AA Wageningen, The Netherlands 2 Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Landscape Dynamics Unit, Zürcherstr. 111, CH-8903 Birmensdorf, Switzerland *Corresponding author; E-mail: [email protected]

ABSTRACT

Wood-anatomical parameters form a valuable archive to study past limitations on tree growth and act as a link between dendrochronology and ecophysiology. Yet, analysing these parameters is a time-consuming procedure and only few long chronologies exist. To increase measurement efficiency of wood-anatomical parameters, novel tools like the automated image-analysis system ROXAS were developed. So far, ROXAS has only been applied to measure large earlywood vessels in ring-porous species. In this study, we evaluate if ROXAS is also suitable for efficient and accurate detection and measurement of vessels in diffuse-porous European beech. To do so, we compared the outcome of ROXAS with that of the established measurement programme Image-Pro Plus in terms of efficiency and accuracy. The two methods differed substantially in efficiency with automatic measurements using ROXAS being 19 times faster than with Image-Pro Plus. Although the procedures led to similar patterns in annual variation of mean vessel area and vessel density, the absolute values differed. Image-Pro Plus measured systematically lower mean vessel areas and higher vessel densities than ROXAS. This was attributed to the species-specific technical settings in ROXAS, leading to more realistic results than those obtained using the default settings in Image-Pro Plus. A shortcoming of ROXAS was, however, that small vessels (